观察受体鼠妊娠和胚胎着床情况,并检测胚胎移植时小鼠子宫内膜中白血病抑制因子(Lif)表达水平,探讨超排卵对小鼠胚胎着床潜能的影响。方法:建立超排周期胚胎和自然周期胚胎移植小鼠模型,比较妊娠率、胚胎着床率的差异及其与Lif蛋白的表达水平之间的关系。结果:超排卵周期受体组的妊娠率(20.00%)和胚胎着床率(8.33%)显著低于自然周期组的妊娠率(55.00%)和胚胎着床率(35.00%)(P＜0.05)。自然周期胚胎和超排周期胚胎受体组内膜中Lif蛋白的表达水平相似(P＞0.05),妊娠受体组Lif蛋白的表达水平显著高于未孕受体组(P＜0.05),但单胎妊娠和多胎妊娠受体组内膜中Lif蛋白的表达水平相似(P＞0.05)。结论:超排卵可能降低胚胎的着床潜能,Lif蛋白的表达水平与胚胎着床有关,但与着床胚胎的数目无比例关系。
OBJECTIVE: To investigate the characteristic and phenotype of ectomesenchymal stem cells of human fetal facial processes and the procedure of spontaneous differentiation to smooth muscle cells. METHODS: The primary ectomesenchymal cells of E 50 human fetal facial processes were isolated by 2.5 g/L trypsin and cultured with DMEM/F 12 with 10(-6) U/L leukemia inhibitor factor(LIF). The morphology and growth rate were observed by inverted microscop. After being withdrawn LIF, the characteristic of cells were identified by immunohistochemistry and RT-PCR. Ultrastructure was observed by transmission electron microscope. RESULTS: The cultured cells displayed monolayer growth and were fibroblast-like with 2-4 processes. The cells were stainely positived for anti-human natural killer cell marker-1, Vimentin, S-100, neuron specific enolase, myoglobin and VIII factor, but negatively for glial fibrillary acidic protein, neural fiblament, alpha-SMA and cytokeratin in immunohistochemistry. Two days after being withdrawn the LIF, cells expressed alpha-SMA in protein and mRNA levels. The cells were rich in muscular filament-like structure and dense bodies under transmission electron microscope. CONCLUSION: Cultured cells are undifferentiated ectomesenchymal stem cells. The cells have the potential for differentiating spontaneously to smooth muscle cell.
目的:探讨胚胎发育不良性神经上皮肿瘤(DNT)的临床、影像及病理学特征、诊断及鉴别诊断.方法:回顾性分析8例胚胎发育不良性神经上皮肿瘤患者的临床和影像学资料,进行光镜和免疫组织化学染色观察,并获得6例的随访资料.结果:胚胎发育不良性神经上皮肿瘤男性7例,女性1例,年龄为5～19岁,平均年龄13岁,5例以癫痫小发作为主要临床表现,病变均位于幕上,以皮层为主,影像学检查均无明显的占位效应及瘤周水肿。肿瘤细胞主要由少突胶质样细胞(OLC)、神经元和星形细胞组成,4例伴有皮质发育不良。免疫组织化学结果为神经元及部分少突胶质样细胞呈嗜铬素A、突触素及S-100阳性表达;少突胶质样细胞呈胶质纤维酸性蛋白(GFAP)阴性表达,而星形细胞呈GFAP阳性表达;Ki-67抗原标记指数lt;1%。结论: 胚胎发育不良性神经上皮肿瘤为WHOⅠ级良性肿瘤,可结合临床、影像及病理学表现明确诊断,预后良好,无需放疗和化疗。
Purpose To investigate the characteristics of intraocular growth of mice embryonic stem cells (ESC) in nude mice. Methods The undifferentiated murine ESC in vitro were transplanted into the eyes of nude mice.Mophological and immunohistochemical examinations were implemented. Results Two to three days after transplantation,yellowish-white granules and masses were seen inside the anterior chamber and vitreous cavity and enlarged gradually.Morphological examination showed that there were undifferentiated cells and differentiated cells in anterior chamber and vitreous cavity.The morphology and alignment of some differentiated cells were similar to those of the retina of nude mice.The cells were highly positive in NSE staining. Conclusion The transplanted ESC could grow in the eyes of nude mice and differentiate into neurons and retina-like structure. (Chin J Ocul Fundus Dis，2000，16：213-284)
Objective To observe the effect of transplantation of embryonic stem cell(ES) on neurological functional recovery of injured spinal cord in adult mouse. Methods The ES cells were cultured and induced in vitro. Fifty C57/BL6J mice were made animal model of semicut mice of T9，10. The ES cellderived neural precursors cells were transplanted into the vertebral canalaround injured spinal cord semi-cut mice. Twenty-eight C57/BL6J mice were randomly divided into three groups: sham operation group(group A,n=9), operation／cell group (group B，n=10), and operation／DMEM group(group C,n=9). RT-PCR analysis, X-gal staining and immunofluorescence were used to observe the cells survival and differentiation in the spinal crod. BBB test was performed to study functional improvement. Results ES cells induced and cultured in vitro displayed clonal growth with circle or ovoid shape and had one or more nucleoli. RT-PCR result showed that the induced ES cells expressed mRNA of Nestin and microtubuleassociated protein, but did not express glial fibrillory acidic protein(GFAP). There was statistically significant difference in BBB scoring between group A and groups B, C after operation (P＜0.01). There was statistically significant difference in BBB scoring at 1, 2 and 4 weeks of operation(P＜0.01), but no statistically significant difference at 6 and 8 weeks of operation between groups B and C(P＞0.05). The X-gal staining results werepositive in group B and negative in groups A and C. The immunoflurescence resultshowed neurofilament green fluor and no expression of GFAP in injured spinal cord region. Conclusion After transplantation, ES cellderived cells can survive, transfer into the injury position, and differentiate into neurons, but spinal cord function has no obvious improvement.
Objective To establ ish a new induction method from human embryonic stem cells (hESCs) differentiating into hepatocyte-l ike cells using an adherent culture system with single-step induction. Methods Undifferentiated hESCs were cultured on Matrigel-coated culture plates for 4 days, hepatic differentiation was initiated at 60%-70% confluence by adding Activin A for 5 days. Then the induction medium was replaced by hepatocyte induction medium (HIM) supplemented with fibroblast growth factor 1 (FGF-1) and bone morphogenetic protein 4 (BMP-4) for another 6 days. Finally, the cells were treated with HIM adding hepatocyte growth factor (HGF) and Oncostatin M (OSM) for 5-7 days. The characteristics of differentiated cells were determined by morphology, immunofluorescence staining, RT-PCR, and Periodicacid- Schiff (PAS) test. Results Differentiated cells treated with Activin A, FGF-1, BMP-4, HGF, and OSM sequentially were morphologically larger and became spherical, oval or polygon. Some cells had 2 or 3 nuclei, suggesting that the cells have a hepatocyte-l ike morphology. Differentiated cells at first induction stage could be stained positive by SOX17 and Forkhead (FOX)A2 after induction by Activin A. Then they turned to be α fetoprotein (AFP) and α1 antitripsin positive cells at second induction stage after induction by FGF-1 and BMP-4. Finally, the differentiated cells treated with HGF and OSM showed PAS possitive for glycogen detection. The differentiated cells at various stages also expressed at early (SOX17, FOXA2, and GATA-4),middle (AFP, albumin, and cytokeratin 18), and mature (alcohol dehydrogenase 1C and Cytochrome P4501B1) stage hepatic genes, respectively. Conclusion Using a simple-step induction method and by suppl ied with cytokines consequently, hESCs can be induced to differentiate into hepatocyte-l ike cells. The differentiation method can provide seed cells for hepatic tissue engineering or cell-therapy.
OBJECTIVE: To observe the effect of nerve growth factor (NGF) and nimodipine (NP) on fetal spinal cord graft in repair of injury of spinal cord. METHODS: A total of 144 adult Wistar rats were included in this study. All were made as the hemi-section cavity injury model at the lumbar enlargement and divided into three groups: fetal spinal cord graft (group Tr), fetal spinal cord graft with NGF (group TN), and fetal spinal cord graft with NGF and NP (group TNN). The intracellular concentration of free ionic calcium was measured at the 4th, 8th, and 24th hour, and superoxidase (SOD) and malondialdehyde (MDA) at 3rd, 6th, 12th, 24th and 72nd hour after operation. RESULTS: After spinal cord was injured, the concentration of MDA and intracellular concentration of free ionic calcium increased and reached to the peak at the 6th and 8th hour respectively, but SOD decreased and at 24th hour to its vale. The MDA was significantly lower in group TN than in group Tr, while the SOD was higher (P lt; 0.05). There was no significant difference on intracellular free ionic calcium concentration between group Tr and TN. The concentration of SOD of group TNN was the highest and the intracellular concentration of free ionic calcium was the lowest in the three groups (P lt; 0.05). The weekly mortality was 33%, 31%, 17% respectively in group Tr, TN and TNN. The mortality of group TNN was significantly lower than the other two groups (P lt; 0.01). CONCLUSION: Although the fetal spinal cord graft is an effective method to repair laboratory spinal cord injury, NGF and ND can interrupt secondary injury and increase survival rate of the host.
Objective To establish a safe, effective, and economic feeder-free culture system which is suitable for the culture of human parthenogenetic embryonic stem cells (hPESCs) in vitro. Methods hPESCs were cultured with mTeSRTMl medium (control group) and human foreskin fibroblasts-conditional medium (hFFs-CM) (experimental group). The growth status of hPESCs in both feeder-free culture systems were observed with inverted microscope. Alkaline phosphatase (ALP) analysis and karyotype analysis were used to study the biological characteristics of hPESCs. The expression of hPESCs pluripotent marker Oct-4 was analyzed by RT-PCR. Differentiation experiment in vivo and in vitro was applied to observe the differentiation potential of hPESCs into three germ layers. Results hPESCs had regular morphology with difficulty in differentiation in both culture systems. No obvious difference was observed in morphology and expansion speed of hPESCs between 2 groups. After subcultured for 15 passages in vitro, hPESCs in 2 groups could maintain normal female diploid karyotype 46, XX and pluripotency. The expression of Oct-4 mRNA was positive in 2 groups. hPESCs in 2 groups could form embryonic body in differentiation experiment in vitro and could develop into teratomas containing three germ layers in nude mice. Conclusion Feeder-free culture system of hFFs-CM can sustain the growth of hPESCs and keep hPESCs undifferentiated state for long. A feeder-free culture system of hPESCs is successfully established, which can support the growth of hPESCs, reduce the contamination from animals, decrease the cost of culture, and satisfy the clinical large-scale application.
ObjectiveTo explore the research progress of the cell sources and related signaling pathways of the wound-induced hair follicle neogenesis (WIHN) in recent years.MethodsThe literature related to WIHN in recent years was reviewed, and the cell sources and molecular mechanism were summarized and discussed.ResultsCurrent research shows that WIHN is a rare regeneration phenomenon in the skin of adult mammals, with multiple cell origins, both hair follicle stem cells and epithelial stem cells around the wound. Its molecular mechanism is complicated, which is regulated by many signaling pathways. Besides, the process is closely related to the immune response, the immunocytes and their related cytokines provide suitable conditions for this process.ConclusionThere are still many unsolved problems on the cellular origins and molecular mechanisms of the WIHN. Further study on the mechanisms will enhance the understanding of adult mammals’ hair follicle regeneration and may provide new strategy for functional healing of the human skin.
Purpose To study the proteins expression of genes related to apoptosis of retinal cells in development of human fetus． Methods Fifty cases of retinas of human fetus aged from 12 to 38 we eks were collected and paraffin embedded sections were made． Immunohistochemical method was used． Results Fas protein was expressed by cells of ganglion cell layer, inner and outer nuclear later, which were just formed on 16th week． It was not expressed until 38th week, Fas(+) staining appeared in layers of retina． Fas-L(+) staining was detected in cells of layers of retina on 26th week and the positive staining located in ganglion cell layer on 32th week． Neuronal fiber layer was Fas-L positive． Bax positive staini ng was detected on 8th week． Bax positive nucleus were observed mainly in GCL and ONL on 16th week． It was in INL on 24th week and in Muuml;ller cells inner terminates on 26th week． After this time, all cells of retina were bax immune ne gative staining． Bcl-2(+) staining appeared in differentiating neuroblastic layer on 16th week． Beginning on 24th week, bcl-2 (+) staining was observed in glial cells of GCL and inner terminates of Muuml;ller cell． Conclusion Apoptosis of developing retinal cell may be Fas/Fas-L independent and bax may be involved in apoptosis of the cells． (Chin J Ocul Fundus Dis, 2001,17:55-57)