黄芪对培养的大鼠搏动心肌细胞感染柯萨奇B_2病毒后保护作用的超微观察

前文已经报道应用电子显微镜观察到培养的新生大鼠搏动心肌细胞感染柯萨奇B_2病毒后,细胞超微结构有明显病变,而中药黄芪对柯萨奇B_2病毒感染培养的大鼠心肌细胞所致心肌酶-乳酸脱氢酶及谷草转氨酶的释放,细胞搏动的停止等均有保护作用(待发表资料)。本     

中草药和铬对心肌细胞生长代谢及其抗病毒的实验研究

选用中草药人参、黄芪、麦冬及微量元素铬于体外培养的人及鼠心肌细胞上，研究药物对细胞生长代谢及其抗病毒作用。实验表明：中草药及铬均有促进细胞生长代 谢、延长细胞存活时间、增强鼠心肌细胞自发缩搏动及提示人心肌细胞抗病毒的作用，其中麦冬、黄芪及铬的作用更明显。     

ERK信号通路对病毒性心肌炎心肌细胞CAR表达的影响

目的:观察细胞外信号调节激酶(Extracellular Regulated Protein Kinases,ERK1/2)信号通路对病毒性心肌炎(Viral Myocarditis,VMC)心肌细胞柯萨奇病毒-腺病毒受体(Coxsackie-adenovirus Receptor,CAR)表达的影响。方法:新生SD大鼠心肌细胞体外培养48 h后随机分为3组,除对照组外均体外接种柯萨奇B3m病毒(Coxsackievirus B,CVB),建立VMC细胞模型。C组:DMEM对照组;V组:CVB3m感染组;U+V组:接种病毒前30 min,给予ERK1/2通路抑制剂U0126(10μmol/L)。各组分别于种毒后12 h、24 h、36 h取心肌细胞,用Western blot法测定ERK1/2活化水平及CAR表达量,并按上述时间点观察各组心肌细胞形态、搏动情况、细胞损伤程度,取培养液测定乳酸脱氢酶(LDH)水平。结果:在接种病毒后12 h,V组与C组相比,P-ERK1/2表达增高(3.25±0.61 vs 0.59±0.09,P0.05),CAR表达增高(1.03±0.17 vs 0.78±0.11,P0.05),逐步出现细胞病变,细胞搏动停止,培养液中LDH水平明显增高(1016.67±67.75 vs 336.34±28.67,P0.05),心肌酶学的升高与镜下心肌细胞损伤程度平行;U+V组与V组相比,P-ERK1/2表达降低(1.66±0.28 vs 3.25±0.61,P0.05),CAR表达明显增高(1.73±0.27 vs 1.03±0.17,P0.05),但细胞损伤却明显减轻,LDH水平明显降低(410.06±13.62 vs 1016.67±67.75,P0.05)。动态观察24 h、36 h,同样出现上述变化趋势。结论:ERK1/2信号转导通路参与心肌细胞感染CVB发生急性损伤的过程,并参与调控CAR的表达。在病毒感染后36 h内,阻断ERK1/2信号通路,CAR表达上调,并未加重心肌细胞损伤。     

黄芪、人参增强人心肌细胞抗病毒感染的实验研究——降低对病毒敏感性及诱生干扰素作用

在培养的人心肌细胞上研究了黄芪、人参抗Cox B-3及Echo-19病毒的作用及诱生干扰素的效应,结果表明两种药物分别作用于人心肌细胞48小时及感染病毒的心肌细胞在含药物的营养液中培养均可降低细胞对两种病毒感染的敏感性,此作用与药物诱导心肌细胞产生IFN有关。     

流行性出血热病毒在各种鼠类巨噬细胞和人外周血白细胞培养中的增殖

本文证实了流行性出血热病毒(EHF)在体外培养的长爪沙鼠、金黄地鼠、Balb/c小鼠腹腔巨噬细胞和人外周血白细胞及单核细胞中的增殖。沙鼠和地鼠腹腔巨噬细胞对EHF病毒的敏感性要高于Vero-E_6细胞,而Balb/c小鼠腹腔巨噬细胞的敏感性却低于其他三种培养的细胞。野鼠型EHF病毒(A_9株)和家鼠型EHF病毒(R_(22)株)在三种鼠腹腔巨噬细胞中的繁殖能力未见明显差异。在人外周血白细胞中,其单核细胞对EHF病毒(A_9株)的敏感性高于淋巴细胞。病毒感染单核细胞后4天的病毒滴度可达10~(-3.0)。以上结果说明EHF病毒在单核巨噬细胞中能繁殖和释放感染性病毒,这表明单核巨噬细胞在鼠、人体内EHF病毒感染的建立及病毒在体内扩散至各器官过程中可能起很重要的作用,即起其靶细胞和扩散媒介的作用。     

人胚心肌细胞培养的初步研究(简报)

本室在乳鼠心肌细胞培养的基础上,最近又成功地培养了人胚心肌细胞。现报告如下。材料和方法妊娠17及18周的胎儿,经水囊引产娩出后,立即在无菌条件下取出心室肌,剪碎,以0.06％胰蛋白酶分次消化成单细胞,用含20％小牛血清的Eagle培养基(MEM)制成10~6细胞/毫升的心肌细胞悬液,进行原代人胚心肌细胞单层培养。在倒置显微镜下观察心肌细胞的搏动及形态,拍摄照片,以及录制心肌细胞搏动的电视录像,并作扫描也子显微镜的观察。     

模拟微重力条件下心肌细胞的体外三维固定化培养

观察心肌细胞体外培养形成三维(3D)组织结构的能力和过程及心肌细胞在模拟微重力状态下的3D固定化培养效果。应用酶消化法从新生的乳鼠心室肌组织获取心肌细胞,以Cytodex3为心肌细胞的3D固定化培养载体,将心肌细胞固定化培养于Spinnerflask中,用扫描电镜观察心肌细胞体外培养形成的3D组织结构;以心肌细胞的代谢效率和细胞搏动强度为观察指标,比较心肌细胞在Spinnerflask及HARV(highaspectratevessel)生物反应器中3D固定化培养的差异。结果显示,心肌细胞不仅能贴附于Cytodex3上生长,且形成了具有同步自律收缩的3D组织样结构;心肌细胞在两种不同培养体系中的细胞接种效率和细胞形态没有明显差异,培养于HARV中的心肌细胞的代谢效率和细胞搏动强度均明显高于Spinnerflask培养体系。体外培养的乳鼠心肌细胞具有形成同步自律收缩的3D组织结构的能力;模拟微重力的培养环境有利于改善心肌细胞3D组织样培养物的代谢和功能 。     

三种滇产鼠尾草注射液抗大鼠心肌缺血及细胞凋亡的实验研究

目的：探讨三种滇产鼠尾草注射液(滇丹参、甘西鼠尾、褐毛甘西鼠尾)对大鼠心肌缺血与细胞凋亡的保护作用。方法：采用大鼠冠脉结扎模型,研究三种滇产鼠尾草注射液对缺血心肌心电图、血清CK、LDH的影响,光镜、电镜下心肌细胞形态学的变化及对细胞凋亡的影响。结果：三种药物均能显著减轻心电图ST段升高,降低心肌酶(CK、LDH)活性,缩小心肌梗塞面积,减轻心肌细胞超微结构损伤,降低心肌细胞凋亡率。结论：三种药物均有抗心肌缺血与细胞凋亡的作用。     

赤芍总苷对培养乳鼠心肌细胞损伤的保护作用

为了探讨赤芍总苷对心肌细胞损伤的保护作用,以异丙基肾上腺素加入培养的乳鼠心肌细胞造成缺血缺氧损伤模型,对比分析正常对照组、药物损伤组、辅酶Q10阳性对照组、以及不同剂量赤芍总苷保护组的细胞形态学、心肌酶谱等指标、结果显示：损伤组细胞搏动加速,存活率下降,GOT、LDH、CK等3种心肌酶活力均显著升高;而辅酶Q10组和赤芍总苷组上述指标都有不同程度的改善,其中高剂量赤芍总苷组的保护作用优于阳性对照组．证明赤芍总苷对异丙基肾上腺素造成的培养乳鼠心肌细胞损伤具有保护作用,并且呈现剂量依赖关系．     

培养心肌细胞肾上腺素能受体的研究

自1960年Harary用胰酶消化分散新生大鼠单个心肌细胞培养成功以后,其他学者相继培养成功大鼠胎鼠和人胚分散心肌细胞。我国学者参照Harary的方法于1981年培养大鼠乳鼠单个心肌细胞成功,有的学者还于1983年观察了β受体药物异丙肾上腺素和心得安对心肌细胞搏动频率的影响。 为了研究单个心肌细胞和其它细胞混合培养时,心肌细胞和其它细胞形态分化和化学分化     

Effects of asbestos on initiation of DNA damage, induction of DNA-strand breaks, P53-expression and apoptosis in primary, SV40-transformed and malignant human mesothelial cells

Human mesothelial cells (HMC), the progenitor cells of asbestos-induced mesothelioma, are particularly sensitive to the genotoxic effects of asbestos, although the molecular mechanisms by which asbestos induces injury in HMC are not well known. The high susceptibility of HMC to simian virus 40 (SV40)-mediated transformation is assumed to play a causative role in the pathogenesis of mesothelioma. The aim of this study was to investigate the asbestos-induced DNA damage in cultured HMC and SV40-transformed HMC (MeT-5A) compared with their malignant counterparts, i.e. human mesothelioma cells (MSTO). The time-dependent initiation of DNA-strand breaks as well as the induction of oxidative DNA base modifications were key factors for investigation. HMC, MeT-5A and MSTO cells were exposed to chrysotile and crocidolite asbestos (3 microg/cm2) during different time periods (1-72 h). DNA damage was investigated by use of the Comet assay and alkaline unwinding, the latter in combination with the Fpg protein. The P53 level was analyzed by immunofluorescence, and measurement of apoptosis was conducted by flow cytometry. We found a significant induction of DNA damage in asbestos-treated HMC already after an exposure time of 1.5 h. This effect could not be observed in treated MeT-5A and MSTO cells. Also, a time-dependent significant increase in DNA-strand breaks was observed by alkaline unwinding in asbestos-treated HMC, but not in treated MeT-5A and MSTO cells. In none of the three cell lines we could detect oxidative DNA damage recognized by the Fpg protein (e.g. 8-oxo-guanine), up to 24 h after exposure to asbestos. In contrast to what was found in HMC, P53 was over-expressed in untreated MeT-5A and MSTO. The induction of apoptosis by asbestos fibers was suppressed in MeT-5A and MSTO cells. Crocidolite fibers induced the higher genotoxic effects and chrysotile the more pronounced apoptotic effects. We conclude that asbestos induces DNA damage in HMC already after a very short exposure time in the absence of 8-oxo-guanine formation. The presence of SV40-Tag in MeT-5A and MSTO cells results in an increased expression of P53, but not in additive genotoxic effects after exposure to asbestos. The deregulation of the apoptotic pathway may lead to proliferation of genomically damaged cells and finally to the development of mesothelioma.     

Increased blastocyst formation of cloned porcine embryos produced with donor cells pre-treated with Xenopus egg extract and/or digitonin

Pre-treating donor cells before somatic cell nuclear transfer (SCNT, 'cloning') may improve the efficiency of the technology. The aim of this study was to evaluate the early development of cloned embryos produced with porcine fibroblasts pre-treated with a permeabilizing agent and extract from Xenopus laevis eggs. In Experiment 1, fetal fibroblasts were permeabilized by digitonin, incubated in egg extract and, after re-sealing of cell membranes, cultured for 3 or 5 days before use as donor cells in handmade cloning (HMC). Controls were produced by HMC with non-treated donor cells. The blastocyst rate for reconstructed embryos increased significantly when digitonin-permeabilized, extract-treated cells were used after 5 days of culture after re-sealing. In Experiment 2, fetal and adult fibroblasts were treated with digitonin alone before re-sealing the cell membranes, then cultured for 3 or 5 days and used as donor cells in HMC. Treatment with digitonin alone increased the blastocyst rate, but only when fetal, and not adult fibroblasts, were used as donor cells, and only after 3 days of culture. In conclusion, we find a time window for increased efficiency of porcine SCNT using donor cells after pre-treatment with permeabilization/re-sealing and Xenopus egg extract. Interestingly, we observe a similar increase in cloning efficiency by permeabilization/re-sealing of donor cells without extract treatment that seems to depend on choice of donor cell type. Thus, pre-treatment of donor cells using permeabilizing treatment followed by re-sealing and in vitro culture for few days could be a simple way to improve the efficiency of porcine cloning.     

Dengue type 2 virus infection in human peripheral blood monocyte cultures

Dengue type 2 virus (D2V) infection in cultured human monocytes was studied. D2V permissiveness of the monocytes was enhanced when the cells were inoculated with D2V in the presence of either polyclonal or type-specific monoclonal anti-dengue antibody. The enhancement of D2V permissiveness mediated by the antibodies was more clearly demonstrated when the monocytes had been treated with trypsin before virus inoculation, though treatment of the cells with trypsin alone decreased D2V permissiveness. The enhancement of infection by type-specific neutralizing monoclonal antibody suggests that the D2V particles possess at least two antigenic determinants closely associated with virus infectivity. Infectious center assays revealed that the infection enhancement in the presence of the antibodies was due primarily to an increase in the number of D2V-infected cells, and that only a small proportion of the monocyte population supported D2V replication. The virus-permissive monocytes did not bear HLA-DR antigens on their cell surface. The presence of nonadherent lymphocytes in the monocyte cultures before D2V inoculation did not affect the D2V permissiveness of the monocytes. Treatment of cultured monocytes with the synthetic adjuvants N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) and its lipophilic derivative, [B30]-MDP, did not significantly affect the D2V permissiveness of the cells.     

Lactate dehydrogenase-elevating agent is responsible for interferon induction and enhancement of natural killer cell activity by inoculation of Ehrlich ascites carcinoma cells into mice

Inoculation of Ehrlich ascites carcinoma cells (EAC) into the peritoneal cavities of outbred ddY mice induced interferon (IFN) in the circulation. The maximum titer (1,280 U) was obtained at 24 hr after inoculation. This induced IFN had the characteristics of type I IFN, i.e., stability at pH2 and lability at 56 C. An increase in natural killer cell (NK) activity was also observed for the first 3 days after inoculation. In addition, plasma lactate dehydrogenase (LDH) activity was elevated in these mice. Inoculation of ascitic fluid or serum of EAC-bearing mice into normal mice increased plasma LDH activity six- to sevenfold over normal levels and elevated activities persisted throughout the life of the mice. These results suggest that the LDH-elevating agent was responsible for IFN induction and for enhancing NK activity. Because lactate dehydrogenase-elevating virus (LDV) can be eliminated from tumor cells by passage in vitro, we attempted to grow EAC in tissue culture for several months and re-examined whether the inoculation of such cells could elevate plasma LDH activity induce IFN and enhance NK activity. The results showed that inoculation of the passaged cells had no effect on these activities in normal mice. Therefore, we concluded that the IFN inducer was LDV which contaminated the EAC and then enhanced the NK activity. N-tropic murine leukemia virus also contaminated EAC, but this virus was not responsible because cultured cells of EAC still shed this virus.     

Long-term treatment of postmenopausal osteoporosis with active vitamin D3, 1-alpha-hydroxycholecalciferol (1 alpha-OHD3) and 1, 24 Dihydroxycholecalciferol (1, 24(OH)2D3)

The effects of active vitamin D3 analogues on radial mineral content (RMC) in postmenopausal osteoporosis were examined. Seventy eight subjects with postmenopausal osteoporosis were divided into 5 groups; Group 1 (n = 23) as the control group and Group 2 (n = 27), Group 3 (n = 8), Group 4 (n = 9) and Group 5 (n = 11) which were given 1 microgram of 1, 24(R) (OH)2D3 per day, 1 microgram of 1, 24(S)(OH)2D3 per day, 0.5 and 1 microgram of 1 alpha-OHD3 per day for 6 to 24 months, respectively. After 3-months administration of these drugs, RMC values were significantly increased in Groups 2 (102.8 +/- 1.8%), 4 (103.9 +/- 3.3%) and 5 (114.2 +/- 3.6%), when compared with the controls (97.9 +/- 2.4%). RMC in Group 3 (97.9 +/- 2.4%) was not significantly different from the control value. The administration of 1 alpha-OHD3 caused in increase in RMC in a dose-related manner. A rapid decrease in RMC was observed after withdrawal of the treatment in Groups 2, 4, and 5. A subsequent increase in RMC was observed after re-administration of 1 alpha-OHD3 and 1, 24(R)(OH)2D3. Serum Ca levels were increased in the group treated with 1, 24(R)(OH)2D3 and were decreased after the discontinuation of 1 alpha-OHD3 administration. Serum A1-P activity was decreased by treatment with 1 alpha-OHD3 (1 microgram per day) and a subsequent increase was observed in both groups treated with 1, 24(R)(OH)2D3 and 1 alpha-OHD3. Serum PTH levels were decreased by the administration of 1, 24(R)(OH)2D3 and 1 alpha-OHD3. In the group treated with 1 microgram of 1 alpha-OHD3 per day, hypercalcemia (2 out of 11 cases and these patients took calcium tablets) and an increase in BUN (1 out of 2 hypercalcemic patients) were observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation of human anaphylatoxin C3a by rat peritoneal mast cells: a role for the secretory granule enzyme chymase and heparin proteoglycan

Purified human C3a was iodinated (125I-C3a) and used to study the interaction of labeled peptide with rat peritoneal mast cells (RMC). Cellular binding of 125I-C3a occurred within 30 sec, followed by a rapid dissociation from the cell. Both the binding of 125I-C3a and the rate of dissociation from the cell were temperature dependent. At 0 degrees C, the binding of 125I-C3a was increased and the rate of dissociation reduced, as compared with 37 degrees C. Once 125I-C3a was exposed to RMC, it lost the ability to rebind to a second batch of RMC. Analysis of the supernatants by trichloroacetic acid (TCA) precipitation and electrophoresis in sodium dodecyl sulfate polyacrylamide gels (SDS PAGE) revealed a decrease in the fraction of 125I precipitable by TCA and the appearance of 125I-C3a cleavage fragments. Pretreatment of RMC with enzyme inhibitors specific for chymotrypsin, but not trypsin, abrogated the degradation of 125I-C3a. Treatment of RMC bearing 125I-C3a with bis (sulfosuccinimidyl) suberate (BS3) covalently cross-linked the 125I-C3a to chymase, the predominant enzyme found in the secretory granules. Antiserum directed against chymase precipitated 125I-C3a from extracts of RMC treated with BS3. Indirect immunofluorescence of RMC by using the IgG fraction of goat anti-rat chymase showed that chymase is present on the surface of unstimulated cells. Neither purified chymase nor heparin proteoglycan alone had any appreciable effect on 125I-C3a, but together they resulted in prompt degradation of the 125I-C3a. Immunoabsorption of RMC sonicates with specific antibody for chymase completely abrogated the ability of these sonicates to degrade 125I-C3a. The results indicate that 125I-C3a binds to RMC and is promptly degraded by chymase in the presence of heparin proteoglycan.     

Cleavage of p21waf1 by proteinase-3, a myeloid-specific serine protease,potentiates cell proliferation

In this study, we present evidence for the critical role of proteinase-3 (PR3) in the proliferation of myeloid cells via the proteolytic regulation of the cyclin-dependent kinase inhibitor p21(waf1). Expression of recombinant PR3 in rat (RBL) or human (HMC1) mast cell lines increased bromodeoxyuridine incorporation and CDK2 activity compared with RBL and HMC1 cells transfected with an enzymatically inactive PR3 mutant (PR3(S203A)) or with human neutrophil elastase. Western blot analysis of p21(waf1) showed an absence of detectable protein, despite normal levels of p21 mRNA. Ectopic overexpression of p21 restored normal levels of p21 in the RBL/PR3/p21 double transfectants and reverted the proliferative effect of PR3. Inhibition of the 26 S proteasome by lactacystin or of caspases by benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone did not inhibit p21 proteolysis. p21 cleavage correlated with PR3 expression in HMC1 cells infected with recombinant adenoviral vector Ad/PR3. During in vitro studies, purified p21 was cleaved by PR3, resulting in a 10-kDa p21 fragment. Employing double immunofluorescence confocal microscopy, subcellular fractionation, and co-immunoprecipitation, we found that PR3 and p21 colocalized in the cytosol. In human neutrophils treated with tumor necrosis factor-alpha, which induces PR3 re-expression, we observed that p21 disappeared and was reversed by Pefabloc, a serine proteinase inhibitor. The physiopathological implications of the cleavage of p21 by PR3 have to be determined.     

低氧预适应对氧糖剥夺损伤SH-SY5Y细胞的保护作用

目的：观察低氧预适应（HPC）对氧糖剥夺（OGD）损伤人神经母细胞瘤细胞（SH-SY5Y）的保护作用,并探讨其可能机制。方法：SH-SY5Y细胞随机分为4组：正常对照组：常规培养,不进行OGD处理;HPC处理组：将神经元放入低氧培养箱内（2% O₂）,30 min后立即取出,再恢复常氧培养,反复5次;OGD组：无糖培养基、低氧培养箱内（1% O₂）处理细胞10 h,然后复氧复糖培养24 h;HPC+OGD处理组：细胞HPC后,行OGD处理。通过形态学观察,MTT比色法检测细胞存活率,乳酸脱氢酶（LDH）漏出量判断细胞损伤的程度,原位末端标记（TUNEL）法检测凋亡水平,Western blot检测Caspase 3、低氧诱导因子1α（HIF-1α）的蛋白表达。结果：HPC可减轻OGD引起的SH-SY5Y细胞凋亡,降低LDH漏出量,明显增加OGD组SH-SY5Y细胞的活力（P<0.05）。Western blot显示HPC+OGD组Cas-pase 3蛋白的表达明显低于OGD组（P<0.05）;HIF-1α蛋白的表达明显高于OGD组（P<0.05）。结论：HPC对体外培养的SH-SY5Y细胞OGD损伤具有保护作用,其机制可能与上调HIF-1α蛋白有关。