Effect of combined treatment with recombinant interleukin-2 and allicin on pancreatic cancer

This study aimed to evaluate the efficacy of combined treatment with recombinant interleukin-2 (rIL-2) and allicin on pancreatic cancer and explore the potential immunological mechanism. A total of 60 C57/BL6 nude mice pancreatic cancer xenograft models were randomized into four groups of 15 mice per group: control group, allicin treatment group, rIL-2 treatment group, combined treatment with allicin and rIL-2 group. Mice in each group were treated with saline, rIL-2, allicin, or combination of rIL-2 and allicin by weekly i.v injection for four weeks. After four weeks of treatment, eyeballs of the mice were extracted and blood was drawn, percentages of CD4+T, CD8+T and NK cell were analyzed by FACS, IFN-γ level was detected by ELISA. One mouse in each group was sacrificed to measure the weight and volume of the tumor and prepared to the paraffin section of tumor tissue. Apoptosis of the tumor cells was analyzed by TUNEL and FACS. Other mice continued to receive treatment, survival period were compared between each group. We observed a significant suppression of xenograft growth and a significant prolonged survival time in the combined treatment with allicin and rIL-2 group (P < 0.05). The most amount of apoptotic cells were observed in the combined therapy group (P < 0.05). The percentages of CD4+T, CD8+T and NK cell and serum IFN-γ level increased significantly in the combined treatment group compared with other groups (P < 0.05). Combined treatment with allicin and rIL-2 resulted in suppression of tumor growth and prolonged survival time possibly through activation of CD4+T, CD8+T and NK cell.     

羟乙基淀粉沉淀(HES)法分离脐血造血干细胞的临床优势分析

目的:探讨羟乙基淀粉沉淀(HES)法分离脐血单个核细胞(MNCs)的效果。方法:采集脐血31份,应用羟乙基淀粉(HES)沉淀飞和密度离心(Ficoll)法分离脐血MNCs,、纯化CD3+细胞、CD14+细胞、CD34+细胞,以台盼蓝拒染法检测细胞活力。结果:HES法MNCs回收率、CFU-GM计数高于Ficoll法(85.29±3.79 VS 47.06±4.61,t=35.67,P0.05;67.31±11.57/l×105MNCs VS28.54±6.39/l×105MNCs,t=16.33,P0.05);HES法分离的MNCs中CD3+、CD14+、CD34+细胞数均高于Ficoll法(t=5.76,t=2.51,t=6.67,P0.05)。结论:HES法分离MNCs可获得较好的回收率,是人脐血干细胞理想的分离方法。     

三叶青黄酮对荷Lewis肺癌小鼠免疫功能及肿瘤组织凋亡相关蛋白的影响

探索三叶青黄酮对荷Lewis肺癌小鼠免疫功能及肿瘤组织凋亡相关蛋白的影响。将60只小鼠随机分为对照组、模型组、三叶青黄酮高、中、低剂量组(100、50、25 mg/mL)各10只。连续干预14天后,检测各组胸腺指数、脾脏指数、移植瘤组织凋亡及凋亡相关蛋白表达,检测外周血调节性T细胞(Treg)比例。结果显示,与模型组比,三叶青黄酮高中剂量组胸腺指数、脾脏指数和移植瘤组织凋亡率升高(P<0.05),凋亡相关蛋白表达升高(P<0.05),外周血CD4+CD25+Foxp3+Treg细胞所占比例降低(P<0.05)。综上,三叶青黄酮可降低荷Lewis肺癌小鼠Treg细胞比例,提高免疫功能,诱导移植瘤组织凋亡。     

CX43 change in LPS preconditioning against apoptosis of mesenchymal stem cells induced by hypoxia and serum deprivation is associated with ERK signaling pathway

This study was designed to investigate the effect and mechanism of lipopolysaccharide (LPS) preconditioning on survival and connexin 43 (CX43) expression in rat bone marrow mesenchymal stem cells (bMSCs) under hypoxia and serum deprivation (Hypoxia/SD) conditions. Whole marrow cells were obtained from the femora and tibiae of SD rats, and bMSCs were isolated by density gradient centrifugation and attachment culture. Surface antigens were determined by FACS before the experiment using antibodies conjugated directly against anti-rat CD34, anti-CD45, anti-CD29, and anti-CD44. Passage 3 bMSCs were used for all experiments. The effect of LPS preconditioning on bMSCs apoptosis in response to Hypoxia/SD was investigated by an Annexin V-FITC/PI binding assay and a mitochondrial membrane potential (△Ψ_m) assay. Cyc-c released into the cytosol from mitochondria and CX43 in bMSCs was determined by Western blot before and after LPS preconditioning. Subsequently, extracellular signal-regulated kinase (ERK) was inhibited with PD98059 to analyze the role of ERK in modulating CX43 expression after LPS preconditioning. The bMSCs surface antigen profiles obtained by flow cytometry were positive for CD29 and CD44 and negative for CD34 and CD45. The Hypoxia/SD conditions induced significant apoptosis of bMSCs. Compared with the Hypoxia/SD group, cells treated with LPS prevented △Ψ_m from falling significantly. LPS inhibited Hypoxia/SD-induced Cyc-c release. These results were consistent with the total analysis of apoptosis of MSCs. Compared with the control group, the level of CX43 expression in the Hypoxia/SD group and LPS + Hypoxia/SD group decreased significantly at each time point. The level of CX43 expression in the Hypoxia/SD group was lower than that in the LPS + Hypoxia/SD group, while the difference was not significant between the PD98059 + LPS + Hypoxia/SD group and the PD98059 + Hypoxia/SD group (P > 0.05). Compared with the LPS + Hypoxia/SD group, CX43 level in the PD98059 + LPS + Hypoxia/SD group and PD98059 + Hypoxia/SD group decreased significantly (P < 0.05). These results demonstrated that Hypoxia/SD conditions could induce apoptosis of bMSCs markedly. Low-dose LPS preconditioning may preserve the mitochondrial function by maintaining the mitochondrial transmembrane potential and inhibiting Cyc-c release in Hypoxia/SD-induced bMSCs apoptosis. LPS preconditioning also had a stabilizing effect on the cell membrane by inhibiting the decrease of CX43, and this modulating mechanism may be related to the ERK signaling pathway.     

Impact of TREM-2 gene silencing on inflammatory response of endotoxin-induced acute lung injury in mice

Acute lung injury (ALI) is one of the critical clinical respiratory diseases, of which infection is the main cause and the first risk factor. This study investigated the impact of triggering receptor of myeloid cells expression (TREM)-2 gene silencing on inflammatory response of endotoxin-induced ALI in mice. Lentivirus-mediated TREM-2-shRNA was transfected into healthy male C57BL/6 mice, and the lipopolysaccharide-induced ALI model was established. The immunohistochemistry, immunofluorescence, fluorescence quantitative PCR, western blot, and ELISA were applied to detect the pathological changes of lung tissue and expressions of TREM-2, tumor necrosis factor-α (TNF-α), and interleukin 10 (IL-10) in bronchoalveolar lavage fluid. The lentivirus group, saline control group, ALI model group, blank control group, and negative control group were set up at the same time. Results found that, in lentivirus group, the pathological change of lung tissue was significantly lighter than ALI model group (P < 0.05), and the expression of TREM-2 was significantly reduced compared with all control groups (P < 0.05). The levels of TNF-α and IL-10 were significantly increased than all control groups (P < 0.05), while above indexes in negative control group and blank control group showed no significant difference with ALI group (P > 0.05). This study indicates that TREM-2 has a protective effect on inflammatory response of endotoxin-induced ALI in mice, which has provided new potential targets for prevention and treatment of ALI.     

Effects of Bone Marrow Mesenchymal Stem Cells on Hematopoietic Recovery and Acute Graft-Versus-Host Disease in Murine Allogeneic Umbilical Cord Blood Transplantation Model

To investigate the effect of bone marrow mesenchymal stem cells (MSC) on hematopoietic recovery and acute graft-versus-host disease (GVHD) in a murine allogeneic umbilical cord blood transplantation (allo-UCBT) model. MSCs were obtained from C57/BL mouse bone marrow. The MSC phenotypes were identified by flow cytometry (FCM), and their ability to differentiate into osteoblasts and adipocytes was tested. Once murine allo-UCBT and aGVHD models were established, mice were divided into five groups: (1) total body irradiation (TBI) group, each mouse receiving 0.3 ml sterile saline infusion after TBI and used as control; (2) UCB group, receiving 2 × 10⁶ umbilical cord blood mononuclear cells (UCB-MNC) after TBI; (3) UCB+MSC group, receiving 2 × 10⁶ UCB-MNC and 2 × 10⁷ MSC after TBI; (4) UCB+SC group, receiving 2 × 10⁶ UCB-MNC and 2 × 10⁶ spleen cells after TBI; and (5) UCB+SC+MSC group, receiving 2 × 10⁶ UCB-MNC, 2 × 10⁷ MSC and 2 × 10⁶ spleen cells after TBI. To evaluate the engraftment of HSC, the white blood cells, red blood cells, and platelets counts were tested at different time points after transplantation, and the ratio of chimerism was identified by FCM. The acute GVHD clinical scores, recipient mice survival, and the histopathological analyses were used to evaluate the effect of MSC on acute GVHD. MSCs were successfully obtained in vitro and FCM analysis showed that these cells are highly positive for CD90.2, CD44, and negative for CD34, CD45, and they are capable to differentiate into osteoblasts and adipocytes after being induced. Compared to UCB group, the UCB+MSC mice had shorter duration of myelosuppression and higher percentage of donor-derived cells which was up to 22.87 ± 4.3 % and the white blood cell (WBC), red blood cell (RBC), and platelet counts started to increase by day 6 after transplantation. Moreover, the average survival time for UCB+MSC mice was 25.0 ± 10.55 days, while for the UCB group it was 15.5 ± 12.50 days. The UCB+SC mice showed fatigue, loss of appetite, weight loss, arched back, and hair ruffling on day 13 post transplantation. Approximately 50 % of mice showed skin ulcers, had diarrhea and other manifestations of acute GVHD, and all mice were died within 20 days. Histopathological analysis confirmed grade II–IV GVHD manifestation. In addition to transient weight loss, some UCB+SC+MSC mice developed arched back, hair ruffling, diarrhea and other manifestations of acute GVHD. The clinical scores in UCB+SC+MSC mice with acute GVHD (grade I–II or without GVHD) were lower than UCB+SC group (P < 0.05). Bone marrow MSCs can promote hematopoietic recovery and decrease the incidence of acute GVHD in murine allo-UCBT model.     

Differential effects of mesenchymal stem cells on a heterogeneous cell population within lung cancer cell lines

Although mesenchymal stem cells (MSCs) promote lung cancer growth in vivo, in vitro studies indicate that they inhibit the proliferation of lung cancer cells. Because malignant tumors contain a heterogeneous cell population with variable capacity for self-renewal, the aim of this study was to determine whether the inconsistencies between in vitro and in vivo studies are a result of differential effects of MSCs on the heterogeneous cell population within lung cancer cell lines. Human MSCs were isolated from the bone marrow, and their cell surface antigen expression and multi-lineage differentiation capacity was examined at passage 10. CD133+ cells were isolated from A549 and H446 cell lines using immunomagnetic separation. The effects of MSCs on the growth and microsphere formation of heterogeneous cell populations within two lung cancer cell lines (A549 and H446) were compared. MSCs inhibited the in vitro proliferation of both cell lines, but significantly accelerated tumor formation and stimulated tumor growth in vivo (P < 0.05). In CD133+ cells isolated from both A549 and H446 cells, co-culture with MSCs for 1–3 days significantly increased their proliferation (P < 0.05). MSCs also significantly increased microsphere formation in both cell lines (P < 0.05). Selective stimulation of CD133+ cell growth may account for the discrepant effects of MSCs on lung cancer progression.     

Probiotic Bacillus amyloliquefaciens SC06 Prevents Bacterial Translocation in Weaned Mice

Probiotic is a preparation containing microorganisms that confers beneficial effect to the host. This work assessed whether oral administration of Bacillus amyloliquefaciens SC06 (Ba) could decrease bacterial translocation in weaned mice. Weaned C57BL/6 were randomly allocated into three groups: group I as the control group, group II were treated with 0.85 % NaCl. Group III was administered with probiotic Ba 1 × 10⁹ CFU/day dissolved in 100 μl of 0.85 % NaCl for 30 days. Mice were then sacrificed, and tissue were cultured to determine bacterial translocation. Meanwhile, splenic CD4⁺T cells, CD8⁺T cells, B cells, and macrophages were analysised by FACS. Our results showed that probiotic Ba significantly reduced bacteria translocation compared with the control group and 0.85 % NaCl group (P < 0.05), lower levels of bacteria were detected in the MLN, liver, spleen, and kidney of mice. Moreover, significant increase in percentage and number of macrophages were observed in the spleen of Ba-treated mice compared with the control and 0.85 % NaCl groups. Together, these data indicated that Ba could decrease bacterial translocation in weaned mice. This effect seems to be correlated with the changes of macrophage numbers.     

Expression of Treg Subsets on Intestinal T Cell Immunity and Endotoxin Translocation in Porcine Sepsis After Severe Burns

In the present study, the changes of the regulatory T cells (Treg) expression, endotoxin translocation, and the relationships in intestinal lymph nodes were investigated in porcine sepsis induced by severe burns. Flow cytometry, western blot, and Tachypleus amebocyte lysate were applied to study after the burn injury model was built. We found that the upregulated Treg expression was negatively related to the CD3⁺CD4⁺/CD3⁺CD8⁺ ratio (r = ?0.832, P < 0.05) after burn injury-induced sepsis. While Treg expression and portal venous plasma endotoxin translocation levels were positively correlated (r = 0.876, P < 0.05) when compared with the control group. Moreover, we detected a transforming of T cell subsets from T helper 1 cells to T helper 2 cells. Therefore, intestinal Treg cells expression exerts immunosuppressive effects on other intestinal T lymphocytes and was closely related to endotoxin translocation in porcine sepsis after severe burns injuries. Above all, the intestinal Treg cells may play an important role in the intestinal immune barrier system after severe burns injuries.     

Effects of Ezetimibe on Endothelial Progenitor Cells and Microparticles in High-Risk Patients

Imbalance on endothelial turnover can predict cardiovascular outcomes. We aimed at evaluating the effects of lipid-modifying therapies on circulating endothelial progenitor cells (EPCs), endothelial microparticles (EMPs), and platelet microparticles (PMPs) in high cardiovascular risk subjects with elevated C-reactive protein (CRP). Sixty-three individuals with coronary heart disease (CHD) or CHD risk equivalent on stable statin therapy, with LDL-cholesterol <100 mg/dL and CRP ≥2.0 mg/L were selected. After a 4-week run-in period with atorvastatin 10 mg, those with persistent CRP ≥2.0 mg/L were randomized to another 4-week treatment period with atorvastatin 40 mg, ezetimibe 10 mg or atorvastatin 40 mg/ezetimibe 10 mg. EPC (CD34⁺/CD133⁺/KDR⁺), EMP (CD51⁺), and PMP (CD42⁺/CD31⁺) were quantified by flow cytometry. Atorvastatin 40 mg and atorvastatin 40 mg/ezetimibe 10 mg reduced LDL-cholesterol (P < 0.001, paired T test, vs. baseline). Combined therapy, but not ezetimibe reduced CRP. CD34⁺/KDR⁺ EPC were reduced after ezetimibe alone (P = 0.011 vs. baseline, Wilcoxon test) or combined with atorvastatin (P = 0.016 vs. baseline, Wilcoxon test). In addition, ezetimibe increased CD51⁺ EMP (P = 0.017 vs. baseline, Wilcoxon test). No correlations between these markers and LDL-cholesterol or CRP were observed. These results contribute to understand the link between inflammation and vascular homeostasis and highlight the broader benefit of statins decreasing inflammation and preventing microparticles release, an effect not observed with ezetimibe alone.     

Immunoregulation Effects of Bone Marrow-Derived Mesenchymal Stem Cells in Xenogeneic Acellular Nerve Grafts Transplant

This study evaluated whether bone marrow-derived mesenchymal stem cells (BM-MSCs) combined with xenogeneic acellular nerve grafts (xANGs) would reduce the inflammation reaction of xANGs transplantation. BM-MSCs were extracted, separated, purified, and cultured from the bone marrow of rats. Then BM-MSCs were seeded into 5 mm xANGs as experimental group, while xANGs group was chosen as control. Subcutaneous implantation and nerve grafts transplantation were done in this study. Walking-track tests, electrophysiological tests, H&E staining, and immunostaining of CD4, CD8, and CD68 of subcutaneous implantations, cytokine concentrations of IL-2, IL-10, IFN-γ and TNF-α in lymphocytes supernatants and serum of the two groups were evaluated. Walking-track tests and electrophysiological tests suggested the group of BM-MSCs with xANGs obtained better results than xANGs group (P < 0.05). H&E staining and immunostaining of CD4, CD8, and CD68 of subcutaneous implantations showed there were less inflammatory cells in the group of BM-MSCs when compared with the xANGs group. The cytokine concentrations of IL-2, IFN-γ, and TNF-α in BM-MSCs group were lower than xANGs group in lymphocytes supernatants and serum (P < 0.05). However, IL-10 concentrations in BM-MSCs group were higher than xANGs group (P < 0.05). xANG with BM-MSCs showed better nerve repair function when compared with xANG group. Furthermore, xANG with BM-MSCs showed less inflammatory reaction which might indicate the reason of its better nerve regeneration.     

Synergistic Actions of Hematopoietic and Mesenchymal Stem/Progenitor Cells in Vascularizing Bioengineered Tissues

Poor angiogenesis is a major road block for tissue repair. The regeneration of virtually all tissues is limited by angiogenesis, given the diffusion of nutrients, oxygen, and waste products is limited to a few hundred micrometers. We postulated that co-transplantation of hematopoietic and mesenchymal stem/progenitor cells improves angiogenesis of tissue repair and hence the outcome of regeneration. In this study, we tested this hypothesis by using bone as a model whose regeneration is impaired unless it is vascularized. Hematopoietic stem/progenitor cells (HSCs) and mesenchymal stem/progenitor cells (MSCs) were isolated from each of three healthy human bone marrow samples and reconstituted in a porous scaffold. MSCs were seeded in micropores of 3D calcium phosphate (CP) scaffolds, followed by infusion of gel-suspended CD34⁺ hematopoietic cells. Co-transplantation of CD34⁺ HSCs and CD34⁻ MSCs in microporous CP scaffolds subcutaneously in the dorsum of immunocompromized mice yielded vascularized tissue. The average vascular number of co-transplanted CD34⁺ and MSC scaffolds was substantially greater than MSC transplantation alone. Human osteocalcin was expressed in the micropores of CP scaffolds and was significantly increased upon co-transplantation of MSCs and CD34⁺ cells. Human nuclear staining revealed the engraftment of transplanted human cells in vascular endothelium upon co-transplantation of MSCs and CD34⁺ cells. Based on additional in vitro results of endothelial differentiation of CD34⁺ cells by vascular endothelial growth factor (VEGF), we adsorbed VEGF with co-transplanted CD34⁺ and MSCs in the microporous CP scaffolds in vivo, and discovered that vascular number and diameter further increased, likely owing to the promotion of endothelial differentiation of CD34⁺ cells by VEGF. Together, co-transplantation of hematopoietic and mesenchymal stem/progenitor cells may improve the regeneration of vascular dependent tissues such as bone, adipose, muscle and dermal grafts, and may have implications in the regeneration of internal organs.     

Cotransplantation of human umbilical cord-derived mesenchymal stem cells and umbilical cord blood-derived CD34+ cells in a rabbit model of myocardial infarction

The objective of the study is to investigate the effect of hypoxic preconditioning on the immunomodulatory properties of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and the effect of cotransplantation of hUC-MSCs and human umbilical cord blood (hUCB)-derived CD34⁺ cells in a rabbit model of myocardial infarction. hUC-MSCs with or without hypoxic preconditioning by cobalt chloride were plated in a 24-well plate, and then cocultured with hUCB-CD34⁺ cells and PBMCs for 96 h at 37 °C in a 5 % CO₂ incubator. For the negative control, hUC-MSCs were omitted. The groups were divided as follows: A1 = HP-MSCs + hUCB-CD34⁺ cells + PBMC, A2 = hUC-MSCs + hUCB-CD34⁺ cells + PBMC, Negative Control = hUCB-CD34⁺ cells + PBMC. Culture supernatants of each group were collected, and the IL-10 and IFN-γ levels were measured by ELISA. A rabbit model of MI was established using a modified Fujita method. The animals were then randomized into three groups and received intramyocardial injections of 0.4 ml of PBS alone (n = 8, PBS group), hUC-MSCs in PBS (n = 8, hUC-MSCs group), or hUC-MSCs + CD34⁺ cells in PBS (n = 8, Cotrans group), at four points in the infarct border zone. Echocardiography was performed at baseline, 4 weeks after MI induction, and 4 weeks after cell transplantation, respectively. Stem cell differentiation and neovascularization in the infracted area were characterized for the presence of cardiac Troponin I (cTnI) and CD31 by immunohistochemical staining, and the extent of myocardial fibrosis was evaluated by hematoxylin and eosin (H&E) and Masson’s trichrome. IFN-γ was 27.00 ± 1.11, 14.20 ± 0.81, and 7.22 ± 0.14 pg/ml, and IL-10 was 31.68 ± 3.08, 61.42 ± 1.08, and 85.85 ± 1.80 pg/ml for the Control, A1 and A2 groups, respectively, which indicated that hUCB-CD34⁺ cells induced immune reaction of peripheral blood mononuclear cells, whereas both hUC-MSCs and HP-MSCs showed an immunosuppressive effect, which, however, was attenuated by hypoxic preconditioning. The Cotrans group had less collagen deposition in the infarcted myocardium and better heart function than the hUC-MSCs or PBS group. The presence of cTnI-positive cells and CD31-positive tubular structures indicated the differentiation of stem cells into cardiomyocytes and neovascularization. The microvessel density was 12.19 ± 3.05/HP for the hUC-MSCs group and 31.63 ± 2.45/HP for the Cotrans group, respectively (P < 0.01). As a conclusion, both hUC-MSCs and HP-MSCs have an immunosuppressive effect on lymphocytes, which, however, can be attenuated by hypoxic preconditioning. Cotransplantation of hUC-MSCs and hUCB-CD34⁺ cells can improve heart function and decrease collagen deposition in post-MI rabbits. Thus, a combined regimen of hUC-MSCs and hUCB-CD34⁺ cells would be more desirable than either cells administered alone. This is most likely due to the increase of cardiomyocytes and enhanced angiogenesis in the infarcted myocardium.     

Improvement of Cloning Efficiency in Minipigs Using Post-thawed Donor Cells Treated with Roscovitine

Massachusetts General Hospital miniature pigs (MGH minipigs) have been established for organ transplantation studies across the homozygous major histocompatibility complex, but cloning efficiency of MGH minipigs is extremely low. This study was designed to increase the productivity of MGH minipigs by nuclear transfer of post-thaw donor cells after 1 h co-incubation with roscovitine. The MGH minipig cells were genetically modified with GT KO (alpha1,3-galactosyltransferase knock-out) and hCD46 KI (human CD46 knock-in) and used as donor cells. The GT KO/hCD46 KI donor cells were cultured for either 3 days (control group) or 1 h after thawing with 15 μM roscovitine (experimental group) prior to the nuclear transfer. The relative percentage of the transgenic donor cells that entered into G0/G1 was 93.7 % (±2.54). This was different from the donor cells cultured for 1 h with the roscovitine-treated group (84.6 % ±4.6) (P < 0.05) and without roscovitine (78.6 % ±5.5) (P < 0.01), respectively. The pregnancy rate and delivery rate in the roscovitine group (8/12 and 6/8, respectively) were significantly higher (P < 0.01) than those in the control group (6/19 and 3/6, respectively). In the experimental group, 12 GT KO/hCD46 KI transgenic minipigs were successfully generated, and five minipigs among them survived for more than 6 months so far. The recipient-based individual cloning efficiency ranged from 0.74 to 2.54 %. In conclusion, gene-modified donor cells can be used for cloning of MGH minipigs if the cells are post-thawed and treated with roscovitine for 1 h prior to nuclear transfer.     

顺铂联合VEGF抗体对食管癌移植瘤小鼠免疫调节以及癌细胞增殖和肺转移的影响

摘要 目的：研究顺铂联合血管内皮生长因子（vascular endothelial growth factor,VEGF）治疗对食管癌移植瘤小鼠免疫功能、癌细胞增殖以及肺转移的影响。方法：30只BALB/c小鼠通过皮下注射食管癌移植瘤模型。一周后,30只食管癌移植瘤模型小鼠被随机均分为3组,即模型组、顺铂组和联合组。模型组不进行治疗,顺铂组腹腔注顺铂治疗,联合组腹腔注射顺铂联合尾静脉注射VEGF抗体进行治疗,共治疗7周。比较各组小鼠体重,食管癌移植瘤体积和重量,卵巢癌细胞肺组织转移结节数、癌细胞转移面积和转移病灶总数,以及食管癌移植瘤外周血CD4⁺、CD8⁺以及CD4⁺/CD8⁺ T淋巴细胞比例。结果：（1）顺博组和联合组小鼠体重均显著高于对照组,而联合组小鼠体重显著高于顺铂组（P<0.05）;（2）顺铂组和联合组小鼠CD4+和CD8+细胞比例均显著低于对照组（P<0.05）,而CD4⁺/CD8⁺却显著高于对照组（P<0.05）;（3）联合组小鼠CD4⁺和CD8⁺细胞比例均显著高于对照组（P<0.05）,而CD4⁺/CD8⁺却显著低于顺铂组（P<0.05）;（4）顺铂组和联合组小鼠食管癌肿瘤组织体积和重量,肺转移结节数、转移面积和转移病灶数均显著低于对照组（P<0.05）,而联合组小鼠显著低于顺铂组（P<0.05）。结论：VEGF抗体可以显著增强顺铂在体内对食管癌的抗癌特性,并有助于增强食管癌移植瘤小鼠免疫功能、抑制癌细胞体内增殖和肺部转移。     

Establishment of Rat Model of Central Venous Catheter (CVC): Associated Infection and Evaluation of the Virulence of Bacterial Biofilms

In this study, a central venous catheter (CVC)—associated infection model was established in rats to investigate and evaluate the effect of biofilms on the virulence of the pathogens. Twenty-four adult SD rats were randomly divided into biofilm positive (BF+) and biofilm negative (BF?) groups to be challenged with strains of S.epidermidis. Serum levels of inflammatory cytokines were measured and the infection rate and counts of bacteria cells were studied. Compared to rats of BF? group, the serum level of TNF and IL-6 significantly increased in rats of BF+ group (P < 0.01) and the level of IL-10 and IFN-γ significantly decreased (P < 0.01), striking the balance of pro-inflammatory/anti-inflammatory cytokines. The infection rate and bacterial counts in tissues and blood of rats of BF + group were significantly higher than those of rats of BF? group (P < 0.05).Inflammatory cell infiltration in vital organs (heart, lung, liver and kidneys) was more significant in rats of BF+ group than that of rats of BF- group. CVC-associated infection model can be successfully reproduced in rats by injecting 5 × 10⁶ CFU of S.epidermidis. Biofilm formation can significantly enhance the virulence of the bacteria, leading to uncontrolled infection. The serum level of inflammatory cytokines, infection rate and the extent of inflammatory cell infiltration are important markers for evaluating the virulence of biofilm.     

Evaluation of Angiogenesis in Non-small Cell Lung Carcinoma by CD34 Immunohistochemistry

The objective of the study was to investigate angiogenesis in non-small lung cancer by measuring the expression of CD34. Immunohistochemistry was used to detect CD34 at the endothelial cell surface in 81 surgically resected non-small cell lung cancer specimens. CD34 immunohistochemistry had high specificity and sensitivity with minimal background, which enabled efficient identification of CD34-positive staining. Statistical analysis showed that increased microvessel density (MVD) was closely correlated to tumor progression as reflected by the clinical stage (P < 0.05). However, MVD was not significantly associated with lymph node metastasis and other clinical and pathological features (P > 0.05). In conclusion, microvascular density may play an important role in the development and progression of lung cancer.     

Transplantation of Immortalized CD34+ and CD34- Adipose-Derived Stem Cells Improve Cardiac Function and Mitigate Systemic Pro-Inflammatory Responses

Adipose-derived stem cells (ADSCs) have the potential to differentiate into various cell lineages and they are easily obtainable from patients, which makes them a promising candidate for cell therapy. However, a drawback is their limited life span during in vitro culture. Therefore, hTERT-immortalized CD34+ and CD34- mouse ADSC lines (mADSCs^hTERT) tagged with GFP were established. We evaluated the proliferation capacity, multi-differentiation potential, and secretory profiles of CD34+ and CD34- mADSCs^hTERT in vitro, as well as their effects on cardiac function and systemic inflammation following transplantation into a rat model of acute myocardial infarction (AMI) to assess whether these cells could be used as a novel cell source for regeneration therapy in the cardiovascular field. CD34+ and CD34- mADSCs^hTERT demonstrated phenotypic characteristics and multi-differentiation potentials similar to those of primary mADSCs. CD34+ mADSCs^hTERT exhibited a higher proliferation ability compared to CD34- mADSCs^hTERT, whereas CD34- mADSCs^hTERT showed a higher osteogenic differentiation potential compared to CD34+ mADSCs^hTERT. Primary mADSCs, CD34+, and CD34- mADSCs^hTERT primarily secreted EGF, TGF-β1, IGF-1, IGF-2, MCP-1, and HGFR. CD34+ mADSCs^hTERT had higher secretion of VEGF and SDF-1 compared to CD34- mADSCs^hTERT. IL-6 secretion was severely reduced in both CD34+ and CD34- mADSCs^hTERT compared to primary mADSCs. Transplantation of CD34+ and CD34- mADSCs^hTERT significantly improved the left ventricular ejection fraction and reduced infarct size compared to AMI-induced rats after 28 days. At 28 days after transplantation, engraftment of CD34+ and CD34- mADSCs^hTERT was confirmed by positive Y chromosome staining, and differentiation of CD34+ and CD34- mADSCs^hTERT into endothelial cells was found in the infarcted myocardium. Significant decreases were observed in circulating IL-6 levels in CD34+ and CD34- mADSCs^hTERT groups compared to the AMI-induced control group. Transplantation of CD34- mADSCs^hTERT significantly reduced circulating MCP-1 levels compared to the AMI control and CD34+ mADSCs^hTERT groups. GFP-tagged CD34+ and CD34- mADSCs^hTERT are valuable resources for cell differentiation studies in vitro as well as for regeneration therapy in vivo.     

Changes in Calcium/Calmodulin Level and Mitochondrial Membrane Potential in Cochlear Neurons of Newborn Mice Infected with Murine Cytomegalovirus

We sought to elucidate the pathogenesis of hearing loss in newborns due to congenital cytomegalovirus. We used the model of murine cytomegalovirus (MCMV) infection and evaluated concentrations of free calcium, calmodulin levels, and mitochondrial membrane potential in cochlear neurons of infected newborn mice. MCMV infection was established by intracranial inoculation of newborn mice with viral suspension (20 μl of MCMV TCID₅₀—10⁴ IU/0.1 ml); the mice in control group were injected 0.9 % NaCl. Concentration of intracellular free calcium concentration ([Ca²⁺] _i ), mitochondrial membrane potential, and the mRNA level of calmodulin (CaM) in the cochlear neurons were evaluated, when the mice were 1 month old. Compared with control group, intracellular [Ca²⁺] _i and CaM mRNA levels significantly (p < 0.05; both comparisons) increased, while the mitochondrial membrane potential significantly (p < 0.05) decreased in the MCMV-infected group. In conclusion, alteration of [Ca²⁺] _i and CaM levels and mitochondrial membrane potentials in cochlear neurons may be the pathological basis of sensorineural hearing loss associated with MCMV infection.     

The Effects and Mechanism of miR-92a and miR-126 on Myocardial Apoptosis in Mouse Ischemia-Reperfusion Model

Our objective was to explore the effects of miR-92a and miR-126 on myocardial apoptosis in mouse ischemia-reperfusion model and further investigate the underlying mechanisms. Eighteen Kunming mice were selected and randomly divided into sham operation group and ischemia-reperfusion group with nine mice in each group. Cardiac muscle tissue was stained with Evans blue to confirm myocardial infarction and ischemia. Annexin V/PI double staining was used to detect the apoptotic rate of myocardial cells, and terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) was used to detect the number of apoptotic cells; Western blot was used to detect expression of Caspase 3 to evaluate the apoptosis of mouse myocardial cells; qRT-PCR was used to detect expression of miR-92a and miR-126 in mouse myocardium, and Western blot was used to detect expression of HSP70 in two groups. Evans blue staining results showed that there was a large area of ischemia in myocardium of ischemia-reperfusion mice with marked infarction, suggesting successful establishment of the model. In sham operation group, myocardial cells were mostly normal cells. Annexin V/PI double staining of flow cytometry result showed that the apoptotic rate was 5.9 % in sham operation group and 37.0 % in ischemia-reperfusion group, respectively. Apoptosis detection results showed that apoptotic index (AI) of myocardial cells in ischemia-reperfusion mice was significantly higher than in sham operation group. In addition, qRT-PCR results showed that miR-92a expression in ischemia-reperfusion group was significantly higher than in sham operation group (F = 32.302, P = 0.000), and miR-126 expression in ischemia-reperfusion group was significantly lower than in sham operation group (F = 41.125, P = 0.000). Moreover, HSP70 detected by Western blot showed that HSP expression in ischemia-reperfusion group was significantly lower than in sham operation group. The change of miR-92a was in accordance with AI of myocardial cells. However, the change of miR-126 is in contrary with AI of myocardial cells, which may be related to the HSP70 expression in myocardial cells.