胎盘脱细胞基质作为组织修复材料的初步研究

目的 制备胎盘脱细胞基质并评价其生物相容性,探讨其作为组织修复材料的可行性。方法 利用分娩废弃物胎盘组织,进行病毒灭活、脱细胞处理、冷冻干燥得到胎盘脱细胞海绵状基质材料,HE染色观察脱细胞效果及扫描电镜观察材料微观结构。同时,选取健康雄性SD大鼠39只,体质量120～150 g,随机分为实验1组、实验2组及对照组。对构建的基质材料进行大鼠皮下植入实验,实验1组植入基质材料,实验2组植入基质材料及脐带间充质干细胞,对照组为假手术组。于手术后第3、5、7天进行动物血常规检测,分析淋巴细胞、粒细胞等炎性细胞数量;于手术后第1、2、4、8、9周取材料植入处及周围组织样本进行HE染色分析。结果 构建的胎盘脱细胞基质肉眼观呈乳白色海绵状,HE染色未见细胞残留,电镜观察材料内部空隙比较明显,材料交联度较好,总孔隙率为（77.54±2.53）%。皮下植入之后,切口处愈合良好,血常规结果未见明显的炎性细胞增多;术后7 d植入材料切片HE染色即可见血管形成,且脐带间充质干细胞的加入能够加快材料与机体的融合,促进细胞的深入生长及血管化。结论 胎盘脱细胞海绵基质材料具有良好的生物相容性,可作为组织工程材料的理想来源。     

Quantitative morphological changes of the uterine gland cells in relation to physiological events during a pregnancy cycle in Glossina morsitans morsitans

Secretory cells of the uterine gland were investigated morphometrically during the second pregnancy cycle in Glossina morsitans morsitans. In early pregnancy when an egg was present in the uterus, morphometric parameters of the nucleus, the cytoplasm and its organelles were markedly low coincident with low physiological activity in the gland cells. This is the period when a proportion of the assimilated amino acids from the digested blood meals are converted to triglyceride in the fat body and stored. During the second half of pregnancy, when a larva was present in the uterus, there was a striking increase of the cytoplasmic and organelle volumes as well as of the surface areas of rough and smooth endoplasmic reticulum. There was also a marked augmentation of triglyceride-like inclusions, and later of secretory vesicles which released their content into enlarging extracellular reservoirs. This indicated a markedly high activity in the gland cells concomittant with the period of rapid synthesis of large amounts of proteins and lipoproteins destined for the growing intra-uterine larva. Mitochondrial parameters also increased, probably to generate adequate energy for synthesis, transport and secretion of nutriments for the larva. After larviposition, a marked reduction of cellular parameters was measured, and the volume density of lysosome-like structures had increased, indicating that larviposition is followed by degradation of large amounts of cellular organelles involved in biosynthetic processes. Morphometrical investigation of the uterine gland cells demonstrated a temporal correlation of the cellular dynamics with physiological events involved in producing a fully developed third-instar larva at the end of a pregnancy cycle.     

Mesenchymal stem cells derived from human placenta suppress allogeneic umbilical cord blood lymphocyte proliferation

Human placenta-derived mononuclear cells （MNC） were isolated by a Percoll density gradient and cultured in mesenchymal stem cell （MSC） maintenance medium. The homogenous layer of adherent cells exhibited a typical fibroblastlike morphology, a large expansive potential, and cell cycle characteristics including a subset of quiescent cells. In vitro differentiation assays showed the tripotential differentiation capacity of these cells toward adipogenic, osteogenic and chondrogenic lineages. Flow cytometry analyses and immunocytochemistry stain showed that placental MSC was a homogeneous cell population devoid of hematopoietic cells, which uniformly expressed CD29, CD44, CD73, CD105, CD166, laminin, fibronectin and vimentin while being negative for expression of CD31, CD34, CD45 and m-smooth muscle actin. Most importantly, immuno-phenotypic analyses demonstrated that these cells expressed class Ⅰ major histocompatibility complex （MHC-I）, but they did not express MHC-Ⅱ molecules. Additionally these cells could suppress umbilical cord blood （UCB） lymphocytes proliferation induced by cellular or nonspecific mitogenic stimuli. This strongly implies that they may have potential application in allograft transplantation. Since placenta and UCB are homogeneous, the MSC derived from human placenta can be transplanted combined with hematopoietic stem cells （HSC） from UCB to reduce the potential graft-versus-host disease （GVHD） in recipients.     

人脐带和胎盘不同层次间充质干细胞的生物学特性分析

间充质干细胞(mesenchymal stem cell,MSC)在再生医学中具有广阔的应用前景,其临床转化应用已成为研究热点,如何大量获取原代间充质干细胞以及针对不同疾病选择最为合适的细胞来源是关键。为了探讨不同来源间充质干细胞的异同,为临床治疗与研究选择合适的种子细胞提供参考,文中比较了人脐带和胎盘不同层次间充质干细胞(MSC)的生物学特性,包括细胞形态、表面标志物、分化能力及核型分析,并对4种胎儿来源的MSCs进行了转录组序列分析,针对转录组测序结果,从增殖能力和分泌细胞因子等方面对不同来源的MSCs进行了分析验证。结果显示,脐带(umbilical cord,UC)、羊膜(amniotic membrane,AM)、绒毛膜平滑肌层(chorionic membrane,CM)、绒毛膜滋养层(chorionicvilli,CV)和蜕膜(deciduae,DC)等5种不同来源的MSCs均符合2006年国际细胞治疗协会(international society for cell therapy,ISCT)的最低标准,符合干细胞的一般特性;核型分析表明除了DC来源MSCs来自母体,UC、AM、CM、CV来源MSCs均来自胎儿;转录组测序分析结果显示,来自脐带和胎盘的MSCs具有相似的基因表达模式,但也存在一些差异,这些特定基因涉及细胞周期、细胞分裂、细胞死亡、细胞生长和发育。这些基因还在转录调节、DNA修复、DNA复制和染色体稳定性中起作用,这是细胞或亚细胞组分运动、细胞通信、细胞组织突起、细胞因子分泌和激素代谢的重要组成部分。转录组测序分析的结果在一定程度上解释了不同来源的MSCs之间生物学特征的差异;基于测序结果的验证实验可知,不同来源的MSCs在增殖能力和细胞因子分泌能力存在显著差异。总的来说,UC-MSCs和CV-MSCs表现出更强的增殖能力,分泌更高水平的旁分泌因子,可能在未来疾病的治疗中有不同的优势。     

Role of apoptosis in functional luteolysis in the pregnant rabbit corpus luteum: Evidence of a role for placental-derived factors in promoting luteal cell survival

Corpora lutea (CL) were isolated from one rabbit ovary on days 4, 8, 16 (peak luteal function), 28 (functional regression) and 30 of pregnancy and processed for biochemical analysis of DNA integrity. Analysis of DNA integrity revealed the presence of oligonucleosomal fragments in day 28 and day 30 CL but not in day 16 CL. The extent of low molecular weight (<15 kb) DNA labeling was 6.6 +/- 0.84 fold higher in day 30 as compared to day 16 CL (mean +/- SEM; n = 4, P < 0.01). In a second series of experiments, healthy CL collected from day 16 pregnant rabbits were incubated for 2 h in the absence or presence of 250 microg/ml of placental extract (PE) obtained from day 16 and/or day 30 placentas. Analysis of DNA integrity revealed that extensive apoptosis occurred in CL incubated in medium alone and in medium containing day 30 PE. In contrast, day 16 PE significantly suppressed apoptosis vs control (70 +/- 4%). In a third series of experiments, expression of mRNA for bcl-x and bax was measured by Northern analysis of CL treated without and with day 16 PE using cRNA probes for bcl-x and bax developed in our laboratory by RT-PCR. Treatment with PE significantly reduced bax mRNA levels but did not change bcl-x mRNA levels. These studies provide evidence that functional luteolysis in the pregnant rabbit CL is correlated with the occurrence of apoptosis. The data suggest that a factor(s) derived from the placenta may be responsible for the prolongation of CL life span during pregnancy by its ability to alter the bax:bcl -x rheostat and suppress apoptosis.     

Stereology of the myocardium in Leontopithecus (Lesson, 1840) callitrichidae - primates

Rare morphological features of the Leontopithecus cardiovascular system have been reported in the literature. The samples analyzed in this study came from 33 specimens of Leontopithecus from the collection of the Center of Primatology of Rio de Janeiro-FEEMA (CPRJ-FEEMA). Morphometry and stereological data were obtained from all animals. Adult body weights of L. rosalia were the lowest, the greatest being those of L. chrysopygus caissara; body weights of L. chrysomelas and L. c. chrysopygus were similar and in between those of the two former species. Cardiomyocytes (left ventricular myocardium) were bigger in adults than in infants. The myocardium of L. rosalia showed focal fibrosis, fatty vacuoles, and hyalinization. In L. chrysomelas the myocardium showed areas of fibrosis and presence of mononuclear cells. Fibrosis and areas of congestion were observed in L. c. chrysopygus; areas of disorganization and vascular congestion were found in L. c. caissara. In L. rosalia infants, a greater density of vessels per myocardial area and a greater length density of vessels were observed as compared with those of L. chrysomelas. In adults, L. chrysomelas showed greater density of connective tissue in the myocardium than L. c. chrysopygus and L. c. caissara did. In L. rosalia, cardiomyocyte nuclei had a greater area density than those of the other forms of Leontopithecus. These characteristics may explain the faster development of L. rosalia infants as compared with that of L. chrysomelas and L. c. chrysopygus kept under the same handling conditions at the CPRJ-FEEMA.     

Human cord blood stem cell paracrine factors activate the survival protein kinase Akt and inhibit death protein kinases JNK and p38 in injured cardiomyocytes

Background aimsWe hypothesized that paracrine factors from human umbilical cord blood mononuclear cells (hUCBC) activate in injured cardiomyocytes the survival protein kinase Akt and limit activation of death protein kinases JNK and p38.MethodsWe treated hUCBC with H₂O₂ and measured growth factors and cytokines secreted by hUCBC. We then treated cardiomyocytes with H₂O₂ for 24 h and measured Akt, JNK and p38 activation by means of Western blots. We also measured myocyte viability and apoptosis with the use of fluorescence-activated cell-sorting cytometry. We then investigated myocytes treated for 24 h with H₂O₂ plus hUCBC and myocytes without hUCBC or H₂O₂. Four million hUCBC were placed in transwells permeable only to hUCBC paracrine factors, and the transwells were placed in flasks with H₂O₂+Dulbecco's modified Eagle's medium or in flasks with myocytes plus H₂O₂+Dulbecco's modified Eagle's medium.ResultshUCBC increased secretion during H₂O₂ of hepatocyte growth factor by 338%, insulin-like growth factor by 200%, interleukin-4 by 200%, vascular endothelial cell growth factor by 192%, placental growth factor by 150%, interleukin-10 by 150% and angiogenin by 121%. H₂O₂ increased myocyte JNK activation by 237% and p38 activation by 60%, decreased myocyte viability by 38% and increased necrosis by 34% (all P < 0.01). hUCBC paracrine factors increased in myocytes with H₂O₂ Akt activation by ≥25%, decreased JNK and p38 activation by >35%, increased viability by >22% and decreased apoptosis by >33% (all P < 0.05). Akt inhibitor API-1 prevented the effects of hUCBC and enhanced H₂O₂ decrease of myocyte viability. Addition of JNK inhibitor SP600125 or p38 inhibitor SB203580 to myocytes plus H₂O₂ prevented H₂O₂ decrease in viability and increased hUCBC beneficial effects.ConclusionsDuring free radical stress, hUCBC paracrine factors activate myocyte Akt, which increases myocyte viability by decreasing activation of death-promoting protein kinases JNK and p38.     

Allelic variation in the erythropoietin receptor gene is associated with uterine capacity and litter size in swine

A single nucleotide polymorphism (SNP; C vs. T) that creates an extra GATA-1 site (T allele) in intron 4 of the swine erythropoietin receptor (EPOR) gene was discovered and a genotyping assay for this SNP was developed. A total of 402 gilts from lines selected either at random (control), for ovulation rate (OR) or for uterine capacity (UC) for 11 generations were unilaterally hysterectomized-ovariectomized (UHO) at 160 days of age, mated at approximately 250 days of age and slaughtered at 105 days of pregnancy. Blood samples and spleens were collected from each foetus and the numbers of corpora lutea (CL) and live foetuses, the weights of each foetus and placenta, and each foetal haematocrit were recorded. In addition, intact gilts from the OR line or from a Yorkshire, Landrace, Duroc, crossbred line (BX) were mated and farrowed. At farrowing, the numbers of fully formed and live piglets were recorded for each litter. Genomic DNA was isolated for both the UHO and intact gilts, from foetuses from the UHO gilts that were heterozygous for the EPOR SNP, and from the boars from the BX line and were then used to determine EPOR SNP genotypes. Only CC and CT gilts were observed in the control, OR and UC selected lines. Presence of the EPOR T allele was associated (P < 0.05) with increased UC in these gilts. The number of heterozygous and homozygous foetuses did not differ within UHO litters, or did EPOR genotype influence foetal haematocrit. In intact gilts from the OR line, litter size was significantly associated (P < 0.05) with EPOR SNP genotype. Finally, results from intact gilts of the BX line, in which both the gilt and the boar genotypes were known, allowed an analysis to determine the effect of the gilt and/or the foetal genotype on litter size. This analysis indicated that the predicted foetal genotype (with gilt genotype as covariate) was associated with litter size (an increase of 2.6 +/- 1.0 piglets born alive predicted for homozygous T litters compared with homozygous C litters, P < 0.01) whereas the effect of the gilt genotype (adjusted for foetal genotype) on litter size was not significant. These results indicate that the EPOR SNP is associated with UC and litter size in two distinct populations and could be useful in increasing litter size in swine that are not limited in OR.     

Expression of G-Protein Subunit α-14 Is Increased in Human Placentas from Preeclamptic Pregnancies

G-proteins mediate cellular function upon interaction with G-protein coupled receptors. Of the 16 mammalian G-protein α subunits identified, G-protein subunit α-11 (GNA11) and -14 (GNA14) have been implicated in modulating hypertension and endothelial function. However, little is known about their expression and roles in human placentas. Here, we examined GNA11 and GNA14 protein expression in first trimester (FT), normal term (NT), and severe preeclamptic (sPE) human placentas as well as in NT human umbilical cords. We found that GNA11 and GNA14 were immunolocalized primarily in trophoblasts, villous stromal cells, and endothelial cells in placentas as well as in endothelial and/or smooth muscle cells of the umbilical cord artery and vein. Western blotting revealed that the GNA14, but not GNA11, protein levels were increased (2.5-2.9 fold; p<0.01) in sPE vs. NT placentas. GNA11 protein was detected only in NT, but not FT, placentas, whereas GNA14 protein levels were increased (7.7-10.6 fold; p<0.01) in NT vs. FT placentas. Thus, GNA11 and GNA14 may mediate the function of several cell types in placentas. Moreover, the high expression of GNA14 in sPE placentas may also imply its importance in sPE pregnancies as in the other hypertension-related disorders.