胎盘间充质干细胞诱导分化为胰岛素分泌细胞及其对糖尿病鼠降糖作用的研究
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摘要
目的:胎盘间充质干细胞(placenta-derived mesenchymal stem cells,PMSCs)是利用间充质干细胞易贴壁生长的特性从胎盘组织中分离培养出来的细胞,在体外可被培养、传代,并具有定向分化为神经细胞、脂肪细胞、成骨细胞的潜能,与骨髓间充质干细胞具有相似的生物学特性,已被作为研究细胞定向分化、多向诱导的“种子细胞”,是未来临床替代治疗和基因治疗的新的干细胞来源,已受到广泛的关注。本研究试图探讨胎盘间充质干细胞在特定的诱导条件下分化为胰岛素分泌细胞,从而起到治疗糖尿病的作用。糖尿病(diabetes mellitus,DM)是危害人类健康的严重疾病,分为Ⅰ型糖尿病和Ⅱ型糖尿病,其发病机理主要是胰腺β细胞受损,不能正常分泌足量的胰岛素,使病人血糖升高并引起多系统损害。这种病人只能依靠终生注射外源性胰岛素来进行治疗,既不方便也会给病人带来痛苦,更为重要的是注射胰岛素并不能阻止糖尿病相关的并发症的发生。所以对于β细胞功能完全丧失的糖尿病患者来说,理想的治疗方法是进行胰腺移植,但目前供源紧张是不易解决的问题,并且需克服移植后的排斥反应,所以胰岛素分泌细胞(insulin-producing cells,IPCs)移植则成为较理想的方法。近10余年来,干细胞的研究取得了重大的突破,干细胞在体外已能分化为多种细胞,如神经细胞、成骨细胞等等,可以将特定的功能细胞移植入体内,进而起到替代治疗各种疾病的作用。因此,近年来,许多学者正在积极寻找能够分化为胰岛素分泌细胞的干细胞来源。目前用于胰岛细胞来源研究的干细胞主要有胚胎干细胞和成体干细胞,后者根据细胞来源的不同又分为脐血干细胞、骨髓干细胞、胰腺干细胞等。从理论上讲,胚胎干细胞(embryonic stem cells,ESCs)是一群较原始的细胞,具有全能干细胞的特点,可分化为外中内三个胚层的细胞,是最具分化潜能的种子细胞,但其易引起肿瘤的发生,并存在伦理问题的争议;骨髓间充质干细胞也是干细胞研究的热点,是目前应用最广泛的干细胞,但骨髓干细胞的数量会因个体的衰老而逐渐减少,分化功能也会随着年龄的增长而减低,并且取材较困难,所以寻找新的干细胞来源迫在眉睫。2002年Kaviani首先证实胎盘组织中含有可以分化的细胞,其后Fukuchi等从成熟胎盘小叶中获得的贴壁细胞可表达数种干细胞标志性基因,Bailo和Yen等人亦从胎盘组织中分离出成纤维细胞样、与骨髓间充质干细胞相似的呈漩涡状生长的细胞,并进一步研究了其形态学特点。国内近年来有多个实验室也成功地分离出胎盘间充质干细胞,并诱导其分化为脂肪细胞、成骨细胞、神经细胞、心肌细胞等,这些研究均说明胎盘间充质干细胞具有多向分化的特性。本研究的目的在于进一步探讨胎盘间充质干细胞的分化潜能,探讨和研究胎盘间充质干细胞体外分离、培养、分化的最佳条件,研究诱导胎盘间充质干细胞分化为胰岛素分泌细胞的可能性与可行性,寻找体外诱导分化的最佳诱导体系,并将诱生的胰岛素分泌细胞移植入糖尿病模型小鼠体内,通过测定血糖和观察一般状况来判断诱生细胞的分泌功能和降血糖功能,为胎盘间充质干细胞的临床应用提供基础研究依据。
方法:1、胎盘间充质干细胞的体外分离培养首先选择足月剖宫产的胎盘,选用胎儿面胎盘组织,以1g/L浓度的Ⅳ型胶原酶消化30分钟,以800转/分离心,获得消化后的细胞沉淀,接种于普通培养基中,培养基内含10%胎牛血清和高糖DMEM。培养条件为37℃,5%CO2、95%饱和湿度,72小时后开始换液,2-3天换液一次,显微镜下观察细胞的生长情况。
2、胎盘间充质干细胞的表型测定取第3代生长旺盛的细胞以EDTA消化后,PBS冲洗2遍,调整细胞密度为1×10~6/100ul,分装于6个离心管中,分别加入鼠抗人单克隆抗体CD19、CD44、CD45、CD34、CD29,流式细胞仪检测其表型。
3、胎盘间充质干细胞诱导分化为成骨细胞取第3代生长状况良好的细胞,以胰蛋白酶将其消化为细胞悬液,以10%小牛血清终止消化后PBS冲洗2遍,接种于诱导培养基中,诱导体系包括10%胎牛血清、高糖DMEM、20mmol/Lβ-甘油磷酸钠、50mg/ml维生素C、10mmol/L地塞米松,置37℃5%CO2孵箱培养,每3-5天换液1次,诱导10天后定量检测硷性磷酸酶活性的变化,并以茜素红染色观察诱导细胞形态和染色情况。
4、胎盘间充质干细胞向胰岛素分泌细胞的诱导分化和检测取第3代生长旺盛的细胞以胰蛋白酶消化后,10%胎牛血清终止消化,PBS洗2遍,悬浮培养于6孔培养液中,加入诱导培养基,诱导体系包含低糖DMEM、1%非必需氨酸、0.1mmol/Lβ-巯基乙醇(β-ME)、1mmol/L谷氨酰胺、5ug/L人硷性成纤维生长因子(bFGF)。诱导培养7天和14天后,采用双硫腙(dithizone,DTZ)染色、免疫细胞化学染色和ELISA等方法检测诱生的胰岛素分泌细胞中胰岛素的生成与分泌情况;用RT-PCR方法检测诱生细胞中PDX-1、Insulin1、Insulin2和Glut2等相关基因mRNA的表达;将培养诱生细胞的上清液从尾静脉注射入小鼠体内,通过检测血糖水平的变化来观察诱生细胞所分泌的胰岛素的降糖活性。
5、胎盘间充质干细胞诱导生成的胰岛素分泌细胞移植对糖尿病小鼠的降糖作用的研究选取纯系Balb/c小鼠,腹腔内注射链脲菌素(streptozotocin,STZ),剂量为200mg/kg,注射后14天,测血糖＞16.7mmol/L诊断为糖尿病,以此建立糖尿病小鼠的模型,建立模型后皮下注射诱导14天的诱生细胞,细胞的数量为1×10~8/鼠,间隔5天后再次注射相同数量的诱生细胞,监测糖尿病鼠的血糖水平和小鼠体重的变化,同时观察小鼠的一般状况,来进一步说明诱生的胰岛素分泌细胞移植对糖尿病鼠的降糖作用。
结果:1、胎盘间充质干细胞的分离培养消化胎盘组织后获得少量的单个核细胞,离心后接种于培养基,大约7-14天左右显微镜下观察发现有贴壁生长的细胞,初起细胞呈不规则形,胞核大而园,胞浆丰富,随着培养时间的延长,细胞变为细长梭形,细胞数量逐渐增多,呈密集型生长,在显微镜下观察表现为漩涡状生长,培养大约4周后细胞可以长满瓶底,胞体细长,胞核清晰,细胞间相互融合,形态类似成纤维样细胞,1周左右可传代1次,并且可稳定传至10代以上。
2、胎盘间充质干细胞的表型流式细胞仪检测结果显示:低表达CD29(1.1%),高表达CD44(98.8%)、CD105(67.4%),不表达CD45(0.7%)、CD34(0.1%)、CD19(0.7%)。
3、胎盘间充质干细胞诱导为成骨细胞诱导10天后诱导组细胞硷性磷酸酶(ALP)活性较对照组高,差异有统计学意义(P＜0.05);成骨诱导后细胞重叠生长,并聚集成团,茜素红染色后可见多个散在分布、大小形态各异的不透明棕红色钙化结节。
4、胎盘间充质干细胞诱导分化为胰岛素分泌细胞①诱导7天后悬浮的细胞逐渐呈团簇状生长,形似“鸟巢”;②诱导14天后双硫腙染色,见鸟巢状细胞团被染成洋红色;③免疫组化染色显示诱生的细胞中有胰岛素特异性免疫反应阳性的细胞;④ELISA法测定结果显示诱生的胰岛素分泌细胞在高糖刺激下可分泌胰岛素;⑤尾静脉注射诱导细胞上清液后30分钟,小鼠的血糖由正常的7.0±0.9 mmol/L降至4.7±0.6 mmol/L;⑥RT-PCR法检测到诱导14天的细胞表达Insulin1、Insulin2、PDX-1、mRNA,不表达Glut2 mRNA。
5、胎盘间充质干细胞诱导生成的胰岛分泌细胞移植治疗糖尿病鼠模型结果显示诱生的细胞皮下注射5天后并未使糖尿病小鼠的血糖明显降低,第二次注射后5天,血糖出现明显的降低,从20.3±0.7mmol/L降至17.3±0.39mmol/L,体重有所增加,糖尿病鼠的一般状况明显改善;至第二次移植后15天,糖尿病鼠的血糖水平复又升高,与移植前的血糖水平比较无明显改变。
结论:1、胎盘组织中存在具有间充质干细胞生物学特性的细胞,能在普通培养基中贴壁生长、传代;
2、胎盘间充质干细胞体外可被诱导分化为成骨细胞;
3、胎盘间充质干细胞在适当的诱导体系中能够分化为胰岛素分泌细胞;
4、胎盘间充质干细胞诱生的胰岛素分泌细胞可移植于糖尿病小鼠体内并能在短期内降低糖尿病小鼠的血糖水平,改善糖尿病小鼠的一般状况,在一定的时间内起到替代治疗的作用。
Objective:Placenta mesenchymal stem cellc(PMSCs) were isolated from placenta tissue in use of the characteristic of mesenchymal stem cell that grows adherently and cultured and passaged in vitro.PMSCs have the potential to differentiate to nerve cell, adipocyte and osteoblast which the characteristics of which are similar to marrow mesenchymal stem cells.And they have been established as seed cells which are used to research the potention to differentiate into all kinde of cells including ectoderm、mesoderm、and endoderm.PMSCs are the source of new stem cells that will be used in future clinical substitution treatment and the gene therapy.This has aroused the widespread interest.This research attempts to discuss the function of placenta mesenchyma stem cells,including isolation、cultivation、differentiation and transplantation into diabetic mice.
Diabetes mellitus(DM) is a serious disease that had affected about 200 million persons in the world can be classified toⅠandⅡ.The mechanism of DM is the damage ofβ-cells of pancrea.β-cells cannot produce and secrete sufficient amounts of active insulin in response to an increased demand for insulin which causes blood glucose elevation and sendary complications associated with diabetes.This kind of patients only could depend upon the life-long injection of exogenous insulin,this is not convenient and ofen failing to match insulin with prevailing blood gloucose concentration and mostly not sufficient for preventing the secondary complications. The ideal treatment is pancreas transplantation.But the lack of cadaveric islets for transplantatirs on determines that researchers must explore alternative source of graft material.So recently many approaches have been taken in the world,such as beta-cell replacement therapy,which is a permanent replacement for the lack of endogenous insulin production.The use of stem cells for generating healthy tissues has the potential to change therapies for diabetes mellius resulting from the destruction of beta-cells.Cell engeering of non-beta cells and selective expansion of stem cells are key potential sources,then transplantation of insulin-producing cells becomes an ideal method.So recently many scholars are seeking the source of stem cells which can differentiate to IPCs.Dudng the past years progress has been made in the definition of new strategies to produce mature pancreatic beta cells.The most significant advance is the use of pluripotent embryonic stem cells(ESCs),serving as a kind of regeneration therapy,because they are not only renewable but also constitute a limitless source of various cells.But they are easy to cause the tumor occurrence and initiate the discussion of ethics problem.Marrow mesenchymal stem cell is also the hot spot and the widest applied at present,but MSCs decreases with aging and are dificult to get,so finding a new source of stem cells is imminent.Kaviani confirmed that placenta tissue contains cells which have the ability to differentiate firstly in 2002, then Fukuchi et al.obtained adherent cells from placenta lobule which can express several kinds of symbolic genes of stem cells.Bailo and Yen et al.isolated fibroblast-like cells that grow swirlly similar to MSCs from placenta tissue,this surveyed its morphology feature furtherly.Recent years,many internal laboratories have isolated and cultured PMSCs successfully and induced them differentiate to nerve cell,adipocyte and osteoblast.These progress suggested that placenta stem cells would be manipulated to differentiate into IPCs which express and secrete insulin.Such information is crucial to assess the feasibility of using PMSCs as a potential source for cell replacement therapy.In our study we would focus on PMSCs and demonstrate that PMSCs can be differentiate into IPCs in vitro which can secret active insulin,and the induced IPCs transplantation may play glucose-reducing effect on diabetes mice.Our results may provide evidence that can be applied as a practical therapeutic strategies for cell transplantation against diabete mellitus using PMSCs.
Methods:
1.Isolated cultivation of PMSCs in vitro:First,we isolated the placenta tissue of Embryo side from full-term c-section and digested them with collagenaseⅣ,then we collected cell precipitation and cultured in medium which contains 10%fetal calf serum and high-glucose Dulbeccos modified eagle medium.The conditions were 37℃,5%CO2、95%saturated humidity,changing the fluid every 2-3 days and observing cell's growth situation.
2.Detection of the phenotype in surface of PMSCs:We got the cells of 3 passages digested by EDTA,flushed by PBS twice.Then we adjusted the number of cells to 1×10~6/100ul,added mouse anti-person monoclonal antibody CD19,CD44,CD45, CD34,CD29 to six centrifuge tubes respectively which are filled with the cells and detected the phenotype by flow cytometry.
3.PMSCs were induced to differentiate into osteoblast:We got the cells of 3 passages which growed well,took trypsase as cell suspension,stopped cells with 10%fetus calf serum,flushed cells twice with PBS,cultured them in induction medium(10%fetus calf serum,high-glucose DMEM,20mmol/L sadiumβ-glycerophosphate,50mg/L vitamin C,10mmol/L dexamethasone),cultured in 5% CO2 hatched box at 37℃,changed the fluid one time every 3-5 days.After 10 days we detected the active change of alkalinity phosphatase,observed the shape of cells and dyeing situation.
4.PMSCs were induced and differentiated into IPCs:We got the cells of 3 passages which growed vigorously digested by trypsas,flushed cells with PBS twice,took the suspension cultured in six-well culture plate in induction medium.The induction system contained high-glucose DMEM,10%fetus calf serum,1%nonessential amino acids,0.1 mmol/Lβ-ME,1mmol/Lglutamine,5ug/LbFGF.
5.Examination of insulin producing cells:After inducted and cultured for 14 days, cells were examinated by these methods:DTZ staining;immunohistochemistrical assay,;insulin detection assay using ELISA;detected the expression of mRNA of PDX-1、Insulin1、Insulin2 and Glut2 by RT-PCR,;detection of activity of secreted insulin.
6.IPCs transplantation on diabetic mice:Pure Balb/c mice were chosen to be injected with streptozotocin in abdominal cavity with the dose of 200mg/kg.14 days later,mouse with the level of blood glucose＞16.7mmol/L were judged as DM.The model of DM mice were established.Cells of induced for 14 days were implanted subcutaneously into the shoulder of diabetic mice with the dose of 1×10~8 per mice in the form of cluster suspension for the first time.Repeated the same operation after 5 days and observed effect of transplantation in blood glucose reducing through detecting blood glucose level and the change of body weight at different time.
Results:
1.Isolated cultivation of PMSCs in vitro Mononuclear cells were harvested after placenta tissue was digested,adhered after about 7-14 days,growed like whirlpool,filled the bottom of the bottle after about 4 weeks.The shape of cells varied from irregularity to fusiform and the body of cell was long and thin like fibroblast.The cells passaged at 1 week for the first time and steadily passaged to the 10th generation.
2.Detection of the phenotype in surface of PMSCs According to the detection of the flow cytometry,cells were lowly positive for CD29(1.1%),high positive for CD44(98.8%) and CD105(67.4%),but negative for CD34(0.1%),CD45 (0.7%) and CD19(0.7%).
3.PMSCs were induced to differentiate into osteoblast After 10 days we found the activity of alkalinity phosphatase in induction group was higher than that of control group,and the disparity had statistical significance.
4.PMSCs were induced and differentiated into IPCs The aerosol cells assumed clump shape to grow gradually and took the form of the bird nest after 7 days induction.
5.Examination of insulin producing cells(1) DTZ staining:the differentiated cells of day 14 after induction were harvested and stained by DTZ,the induced cells were found to be stained crimson red.(2)Immunohistoochemiistrical assay for insulin:we examined insulin immunoreactivity within the cluster on the day of 14 after induction. Immunoreactivity was observed in some induced cell clusters.(3) The results of ELISA showed that inducted IPCs could secrete insulin under the stimulation of high glucose.(4) Detection of activity of secreted insulin:The supematant of day 14 could reduce blood glucose significantly when injected into normal mouse and the level of blood glucose decreased from 7.0±0.9 mmol/Lto4.7±0.6 mmol/L 30 min after tail intravenous injection.(5) Gene expression of IPCs cluster:to clarify the characteristical features of the differentiated clusters,we analyzed the gene expression of avariety of endocrine pancreatic markers using RT-PCR analysis.RNA samples were obtained from the induced cells on day 7 and 14.Samples of day 7 expressed PDX-1 mRNA of pancreas specific gene.Samples of day 14 expressed Insulin1、Insulin2、PDX-1mRNA.No expression of Glut2 was observed in both of the two sample.
6.IPCs transplantation on diabetic mice G1 represents blood glucose level before STZ injection.G2 is blood glucose level before IPCs transplantation.G3 is blood glucose level on 5 after the first transplantation.G4and G5 are blood glucose levels of day 5 and day 15 after the second IPCs tiansplantation respectivedly.The results showed that:Blood glucose level of IPCs transplantation group and DM model group before transplantation was increased significantly comparing with that before STZ induction.Blood glucose level of IPCs transplantation group reduced significantly on day 5 after the second transplantation comparing with that before IPCs transplantation,from 20.3±0.7mmol/Lto 17.3±0.39mmol/L,which implied that IPCs transplantation in diabetic mice was effective.Blood glucose level of DM model control and normal control were not changed during the period of experiment.The level of blood glucose had not obvious difference on the day 15 after the second transplantation.
Conclusion:
1.Cells that had the biological characteristics of mesenchymal stem cells exist in placenta tissue and can grow adherently and passage in the common medium.
2.PMSCs could be differentiated into osteoblast.
3.PMSCs could be differentiated into IPCs in proper induction system.
4.The transplantation of insulin-producing cells inducted from PMSCs to DM mice can make the level of blood glucose reduced in a short period and improve general condition of the mice---playing the effect of substituted treatment.
方法:1、胎盘间充质干细胞的体外分离培养首先选择足月剖宫产的胎盘,选用胎儿面胎盘组织,以1g/L浓度的Ⅳ型胶原酶消化30分钟,以800转/分离心,获得消化后的细胞沉淀,接种于普通培养基中,培养基内含10%胎牛血清和高糖DMEM。培养条件为37℃,5%CO2、95%饱和湿度,72小时后开始换液,2-3天换液一次,显微镜下观察细胞的生长情况。
2、胎盘间充质干细胞的表型测定取第3代生长旺盛的细胞以EDTA消化后,PBS冲洗2遍,调整细胞密度为1×10~6/100ul,分装于6个离心管中,分别加入鼠抗人单克隆抗体CD19、CD44、CD45、CD34、CD29,流式细胞仪检测其表型。
3、胎盘间充质干细胞诱导分化为成骨细胞取第3代生长状况良好的细胞,以胰蛋白酶将其消化为细胞悬液,以10%小牛血清终止消化后PBS冲洗2遍,接种于诱导培养基中,诱导体系包括10%胎牛血清、高糖DMEM、20mmol/Lβ-甘油磷酸钠、50mg/ml维生素C、10mmol/L地塞米松,置37℃5%CO2孵箱培养,每3-5天换液1次,诱导10天后定量检测硷性磷酸酶活性的变化,并以茜素红染色观察诱导细胞形态和染色情况。
4、胎盘间充质干细胞向胰岛素分泌细胞的诱导分化和检测取第3代生长旺盛的细胞以胰蛋白酶消化后,10%胎牛血清终止消化,PBS洗2遍,悬浮培养于6孔培养液中,加入诱导培养基,诱导体系包含低糖DMEM、1%非必需氨酸、0.1mmol/Lβ-巯基乙醇(β-ME)、1mmol/L谷氨酰胺、5ug/L人硷性成纤维生长因子(bFGF)。诱导培养7天和14天后,采用双硫腙(dithizone,DTZ)染色、免疫细胞化学染色和ELISA等方法检测诱生的胰岛素分泌细胞中胰岛素的生成与分泌情况;用RT-PCR方法检测诱生细胞中PDX-1、Insulin1、Insulin2和Glut2等相关基因mRNA的表达;将培养诱生细胞的上清液从尾静脉注射入小鼠体内,通过检测血糖水平的变化来观察诱生细胞所分泌的胰岛素的降糖活性。
5、胎盘间充质干细胞诱导生成的胰岛素分泌细胞移植对糖尿病小鼠的降糖作用的研究选取纯系Balb/c小鼠,腹腔内注射链脲菌素(streptozotocin,STZ),剂量为200mg/kg,注射后14天,测血糖＞16.7mmol/L诊断为糖尿病,以此建立糖尿病小鼠的模型,建立模型后皮下注射诱导14天的诱生细胞,细胞的数量为1×10~8/鼠,间隔5天后再次注射相同数量的诱生细胞,监测糖尿病鼠的血糖水平和小鼠体重的变化,同时观察小鼠的一般状况,来进一步说明诱生的胰岛素分泌细胞移植对糖尿病鼠的降糖作用。
结果:1、胎盘间充质干细胞的分离培养消化胎盘组织后获得少量的单个核细胞,离心后接种于培养基,大约7-14天左右显微镜下观察发现有贴壁生长的细胞,初起细胞呈不规则形,胞核大而园,胞浆丰富,随着培养时间的延长,细胞变为细长梭形,细胞数量逐渐增多,呈密集型生长,在显微镜下观察表现为漩涡状生长,培养大约4周后细胞可以长满瓶底,胞体细长,胞核清晰,细胞间相互融合,形态类似成纤维样细胞,1周左右可传代1次,并且可稳定传至10代以上。
2、胎盘间充质干细胞的表型流式细胞仪检测结果显示:低表达CD29(1.1%),高表达CD44(98.8%)、CD105(67.4%),不表达CD45(0.7%)、CD34(0.1%)、CD19(0.7%)。
3、胎盘间充质干细胞诱导为成骨细胞诱导10天后诱导组细胞硷性磷酸酶(ALP)活性较对照组高,差异有统计学意义(P＜0.05);成骨诱导后细胞重叠生长,并聚集成团,茜素红染色后可见多个散在分布、大小形态各异的不透明棕红色钙化结节。
4、胎盘间充质干细胞诱导分化为胰岛素分泌细胞①诱导7天后悬浮的细胞逐渐呈团簇状生长,形似“鸟巢”;②诱导14天后双硫腙染色,见鸟巢状细胞团被染成洋红色;③免疫组化染色显示诱生的细胞中有胰岛素特异性免疫反应阳性的细胞;④ELISA法测定结果显示诱生的胰岛素分泌细胞在高糖刺激下可分泌胰岛素;⑤尾静脉注射诱导细胞上清液后30分钟,小鼠的血糖由正常的7.0±0.9 mmol/L降至4.7±0.6 mmol/L;⑥RT-PCR法检测到诱导14天的细胞表达Insulin1、Insulin2、PDX-1、mRNA,不表达Glut2 mRNA。
5、胎盘间充质干细胞诱导生成的胰岛分泌细胞移植治疗糖尿病鼠模型结果显示诱生的细胞皮下注射5天后并未使糖尿病小鼠的血糖明显降低,第二次注射后5天,血糖出现明显的降低,从20.3±0.7mmol/L降至17.3±0.39mmol/L,体重有所增加,糖尿病鼠的一般状况明显改善;至第二次移植后15天,糖尿病鼠的血糖水平复又升高,与移植前的血糖水平比较无明显改变。
结论:1、胎盘组织中存在具有间充质干细胞生物学特性的细胞,能在普通培养基中贴壁生长、传代;
2、胎盘间充质干细胞体外可被诱导分化为成骨细胞;
3、胎盘间充质干细胞在适当的诱导体系中能够分化为胰岛素分泌细胞;
4、胎盘间充质干细胞诱生的胰岛素分泌细胞可移植于糖尿病小鼠体内并能在短期内降低糖尿病小鼠的血糖水平,改善糖尿病小鼠的一般状况,在一定的时间内起到替代治疗的作用。
Objective:Placenta mesenchymal stem cellc(PMSCs) were isolated from placenta tissue in use of the characteristic of mesenchymal stem cell that grows adherently and cultured and passaged in vitro.PMSCs have the potential to differentiate to nerve cell, adipocyte and osteoblast which the characteristics of which are similar to marrow mesenchymal stem cells.And they have been established as seed cells which are used to research the potention to differentiate into all kinde of cells including ectoderm、mesoderm、and endoderm.PMSCs are the source of new stem cells that will be used in future clinical substitution treatment and the gene therapy.This has aroused the widespread interest.This research attempts to discuss the function of placenta mesenchyma stem cells,including isolation、cultivation、differentiation and transplantation into diabetic mice.
Diabetes mellitus(DM) is a serious disease that had affected about 200 million persons in the world can be classified toⅠandⅡ.The mechanism of DM is the damage ofβ-cells of pancrea.β-cells cannot produce and secrete sufficient amounts of active insulin in response to an increased demand for insulin which causes blood glucose elevation and sendary complications associated with diabetes.This kind of patients only could depend upon the life-long injection of exogenous insulin,this is not convenient and ofen failing to match insulin with prevailing blood gloucose concentration and mostly not sufficient for preventing the secondary complications. The ideal treatment is pancreas transplantation.But the lack of cadaveric islets for transplantatirs on determines that researchers must explore alternative source of graft material.So recently many approaches have been taken in the world,such as beta-cell replacement therapy,which is a permanent replacement for the lack of endogenous insulin production.The use of stem cells for generating healthy tissues has the potential to change therapies for diabetes mellius resulting from the destruction of beta-cells.Cell engeering of non-beta cells and selective expansion of stem cells are key potential sources,then transplantation of insulin-producing cells becomes an ideal method.So recently many scholars are seeking the source of stem cells which can differentiate to IPCs.Dudng the past years progress has been made in the definition of new strategies to produce mature pancreatic beta cells.The most significant advance is the use of pluripotent embryonic stem cells(ESCs),serving as a kind of regeneration therapy,because they are not only renewable but also constitute a limitless source of various cells.But they are easy to cause the tumor occurrence and initiate the discussion of ethics problem.Marrow mesenchymal stem cell is also the hot spot and the widest applied at present,but MSCs decreases with aging and are dificult to get,so finding a new source of stem cells is imminent.Kaviani confirmed that placenta tissue contains cells which have the ability to differentiate firstly in 2002, then Fukuchi et al.obtained adherent cells from placenta lobule which can express several kinds of symbolic genes of stem cells.Bailo and Yen et al.isolated fibroblast-like cells that grow swirlly similar to MSCs from placenta tissue,this surveyed its morphology feature furtherly.Recent years,many internal laboratories have isolated and cultured PMSCs successfully and induced them differentiate to nerve cell,adipocyte and osteoblast.These progress suggested that placenta stem cells would be manipulated to differentiate into IPCs which express and secrete insulin.Such information is crucial to assess the feasibility of using PMSCs as a potential source for cell replacement therapy.In our study we would focus on PMSCs and demonstrate that PMSCs can be differentiate into IPCs in vitro which can secret active insulin,and the induced IPCs transplantation may play glucose-reducing effect on diabetes mice.Our results may provide evidence that can be applied as a practical therapeutic strategies for cell transplantation against diabete mellitus using PMSCs.
Methods:
1.Isolated cultivation of PMSCs in vitro:First,we isolated the placenta tissue of Embryo side from full-term c-section and digested them with collagenaseⅣ,then we collected cell precipitation and cultured in medium which contains 10%fetal calf serum and high-glucose Dulbeccos modified eagle medium.The conditions were 37℃,5%CO2、95%saturated humidity,changing the fluid every 2-3 days and observing cell's growth situation.
2.Detection of the phenotype in surface of PMSCs:We got the cells of 3 passages digested by EDTA,flushed by PBS twice.Then we adjusted the number of cells to 1×10~6/100ul,added mouse anti-person monoclonal antibody CD19,CD44,CD45, CD34,CD29 to six centrifuge tubes respectively which are filled with the cells and detected the phenotype by flow cytometry.
3.PMSCs were induced to differentiate into osteoblast:We got the cells of 3 passages which growed well,took trypsase as cell suspension,stopped cells with 10%fetus calf serum,flushed cells twice with PBS,cultured them in induction medium(10%fetus calf serum,high-glucose DMEM,20mmol/L sadiumβ-glycerophosphate,50mg/L vitamin C,10mmol/L dexamethasone),cultured in 5% CO2 hatched box at 37℃,changed the fluid one time every 3-5 days.After 10 days we detected the active change of alkalinity phosphatase,observed the shape of cells and dyeing situation.
4.PMSCs were induced and differentiated into IPCs:We got the cells of 3 passages which growed vigorously digested by trypsas,flushed cells with PBS twice,took the suspension cultured in six-well culture plate in induction medium.The induction system contained high-glucose DMEM,10%fetus calf serum,1%nonessential amino acids,0.1 mmol/Lβ-ME,1mmol/Lglutamine,5ug/LbFGF.
5.Examination of insulin producing cells:After inducted and cultured for 14 days, cells were examinated by these methods:DTZ staining;immunohistochemistrical assay,;insulin detection assay using ELISA;detected the expression of mRNA of PDX-1、Insulin1、Insulin2 and Glut2 by RT-PCR,;detection of activity of secreted insulin.
6.IPCs transplantation on diabetic mice:Pure Balb/c mice were chosen to be injected with streptozotocin in abdominal cavity with the dose of 200mg/kg.14 days later,mouse with the level of blood glucose＞16.7mmol/L were judged as DM.The model of DM mice were established.Cells of induced for 14 days were implanted subcutaneously into the shoulder of diabetic mice with the dose of 1×10~8 per mice in the form of cluster suspension for the first time.Repeated the same operation after 5 days and observed effect of transplantation in blood glucose reducing through detecting blood glucose level and the change of body weight at different time.
Results:
1.Isolated cultivation of PMSCs in vitro Mononuclear cells were harvested after placenta tissue was digested,adhered after about 7-14 days,growed like whirlpool,filled the bottom of the bottle after about 4 weeks.The shape of cells varied from irregularity to fusiform and the body of cell was long and thin like fibroblast.The cells passaged at 1 week for the first time and steadily passaged to the 10th generation.
2.Detection of the phenotype in surface of PMSCs According to the detection of the flow cytometry,cells were lowly positive for CD29(1.1%),high positive for CD44(98.8%) and CD105(67.4%),but negative for CD34(0.1%),CD45 (0.7%) and CD19(0.7%).
3.PMSCs were induced to differentiate into osteoblast After 10 days we found the activity of alkalinity phosphatase in induction group was higher than that of control group,and the disparity had statistical significance.
4.PMSCs were induced and differentiated into IPCs The aerosol cells assumed clump shape to grow gradually and took the form of the bird nest after 7 days induction.
5.Examination of insulin producing cells(1) DTZ staining:the differentiated cells of day 14 after induction were harvested and stained by DTZ,the induced cells were found to be stained crimson red.(2)Immunohistoochemiistrical assay for insulin:we examined insulin immunoreactivity within the cluster on the day of 14 after induction. Immunoreactivity was observed in some induced cell clusters.(3) The results of ELISA showed that inducted IPCs could secrete insulin under the stimulation of high glucose.(4) Detection of activity of secreted insulin:The supematant of day 14 could reduce blood glucose significantly when injected into normal mouse and the level of blood glucose decreased from 7.0±0.9 mmol/Lto4.7±0.6 mmol/L 30 min after tail intravenous injection.(5) Gene expression of IPCs cluster:to clarify the characteristical features of the differentiated clusters,we analyzed the gene expression of avariety of endocrine pancreatic markers using RT-PCR analysis.RNA samples were obtained from the induced cells on day 7 and 14.Samples of day 7 expressed PDX-1 mRNA of pancreas specific gene.Samples of day 14 expressed Insulin1、Insulin2、PDX-1mRNA.No expression of Glut2 was observed in both of the two sample.
6.IPCs transplantation on diabetic mice G1 represents blood glucose level before STZ injection.G2 is blood glucose level before IPCs transplantation.G3 is blood glucose level on 5 after the first transplantation.G4and G5 are blood glucose levels of day 5 and day 15 after the second IPCs tiansplantation respectivedly.The results showed that:Blood glucose level of IPCs transplantation group and DM model group before transplantation was increased significantly comparing with that before STZ induction.Blood glucose level of IPCs transplantation group reduced significantly on day 5 after the second transplantation comparing with that before IPCs transplantation,from 20.3±0.7mmol/Lto 17.3±0.39mmol/L,which implied that IPCs transplantation in diabetic mice was effective.Blood glucose level of DM model control and normal control were not changed during the period of experiment.The level of blood glucose had not obvious difference on the day 15 after the second transplantation.
Conclusion:
1.Cells that had the biological characteristics of mesenchymal stem cells exist in placenta tissue and can grow adherently and passage in the common medium.
2.PMSCs could be differentiated into osteoblast.
3.PMSCs could be differentiated into IPCs in proper induction system.
4.The transplantation of insulin-producing cells inducted from PMSCs to DM mice can make the level of blood glucose reduced in a short period and improve general condition of the mice---playing the effect of substituted treatment.
引文
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