体外脐血间充质干细胞的培养及其向肝细胞样细胞诱导分化的初步研究

发布时间：2018-09-12 10:58

【摘要】： 目的:从脐血中提取间充质干细胞,进行体外扩增,摸索脐血间充质干细胞在体外的适宜培养环境。探讨间充质干细胞在体外分化为肝细胞的可能性和适宜条件。方法: 1.待胎儿娩出断脐后,无菌条件下采血,并用肝素抗凝。使用相对密度为1.077的ficoll淋巴细胞分离液,利用密度梯度离心原理分离脐血单个核细胞。 2.贴壁培养法,比较不同接种密度、不同血清浓度条件下,间充质干细胞的贴壁情况。从而摸索脐血来源间充质干细胞的适宜培养环境。 3.收集4代的细胞,用流式细胞技术检测细胞表面CD29、CD44、CD34、CD45的表达情况,来鉴定细胞表面的标志物。 4.常规方法冻存和复苏间充质干细胞,观察复苏后细胞的生长情况。 5.收集4-8代的细胞,分成5个诱导组。分别采用与正常肝细胞株LO2分层共同培养法、细胞因子联合诱导法及细胞因子阶段诱导法,观察细胞形态的变化。 6.采用RT-PCR,检测白蛋白(ALB)、甲胎蛋白(AFP)及角质蛋白(CK19)mRNA的表达情况,来鉴定间充质干细胞的诱导分化情况。结果: 1.10~8/ml的接种密度,7d后首次换液,可获得较大密度的间充质干细胞,实现体外生长增殖。 2.5%的血清,细胞不易贴壁、增殖缓慢。10%-15%的血清,有利于干细胞的生长增殖,且不易发生形态变化。20%的血清,细胞生长快,但易发生形态改变。 3.流式细胞技术分析:CD29(+)、CD44(+)、CD34(-)、CD45(-),与间充质干细胞表面标志物的的特点一致。RT-PCR分析:间充质干细胞不表达ALB、AFP及CK19。 4.本实验所获得的间充质干细胞体外传8代后,细胞增殖能力明显减弱,体积变大,呈不规则形。第10代时,无法继续传代,细胞死亡。复苏后,间充质干细胞要经历1w的平台期后,才能逐渐恢复冻存前的状态。 5.第1诱导组(与L02共同培养)、第2诱导组(A组诱导液)、第3诱导组(与LO2共同培养+A组诱导液)诱导6w,较对照组未见形态学变化,也未检测到ALB、AFP及CK19mRNA的表达。 6.第4诱导组(B组诱导液)诱导6w,细胞形态也未有改变,未检测到ALB、AFP及CK19mRNA的表达。 7.第5诱导组(C组诱导液)诱导4w,细胞由长梭型变为多角形,RT-PCR检测到AFPmRNA的阳性表达,继续诱导2w后,检测到AFPmRNA、ALBmRNA、CK19mRNA的阳性表达。但细胞死亡较多,增殖能力差,细胞密度减少明显。结论: 1.脐血中含有间充质干细胞,但含量少,不同个体之间也存在生物学差异。脐血MSCs贴壁较骨髓MSCs晚,体外培养困难。要想实现体外扩增,需要高密度接种、较高的血清浓度,以10-15%为宜。但血清浓度也不能过高,过高的血清浓度(＞20%)促进细胞分化,增殖能力明显降低。 2.体外培养的MSCs,传代能力有限,P4-P8代细胞活力好。P10代细胞死亡,目前尚无法实现MSCs的体外无限增殖。冻存后的MSCs,复苏后,生长缓慢,需要1w的平台期,才能恢复冻存前的状态。 3.体外诱导脐血MSCs向肝细胞分化,非常困难。本实验,5个诱导组,只有第5组阶段诱导法,得到了多角形的肝细胞样细胞,RT-PCR在此种细胞内检测到AFPmRNA、ALBmRNA、CK19mRNA的阳性表达。证明,此种诱导方案是一种可行的诱导方法。 4.诱导过程中,细胞贴壁能力差,活力弱,扩增困难。如何实现诱导后细胞的体外扩增,从而能够应用临床是我们下一步要解决的问题。
[Abstract]:AIM: To extract mesenchymal stem cells from umbilical cord blood and to explore the suitable culture environment for umbilical cord blood mesenchymal stem cells in vitro.
Method:
1. After the umbilical cord was cut off, the blood was collected under aseptic condition and anticoagulated with heparin. The umbilical cord blood mononuclear cells were separated by density gradient centrifugation using Ficoll lymphocyte isolation solution with relative density of 1.077.
2. Comparing the adherence of mesenchymal stem cells with different density and serum concentration, so as to explore the suitable culture environment of umbilical cord blood-derived mesenchymal stem cells.
3. The expression of CD29, CD44, CD34 and CD45 on the cell surface was detected by flow cytometry to identify the markers on the cell surface.
4. cryopreservation and resuscitation of mesenchymal stem cells were used to observe the growth of cells after resuscitation.
5. Cells of 4-8 generations were collected and divided into 5 induction groups. Morphological changes were observed by co-culture with LO2, cytokine induction and cytokine stage induction.
6. The expression of albumin (ALB), alpha-fetoprotein (AFP) and keratin (CK19) mRNA was detected by RT-PCR to identify the differentiation of mesenchymal stem cells.
Result:
At the density of 1.10~8/ml, the mesenchymal stem cells with higher density could be obtained after the first liquid exchange 7 days, and the proliferation of mesenchymal stem cells could be realized in vitro.
In 2.5% serum, the cells are not easy to adhere to the wall and proliferate slowly. 10% - 15% serum is conducive to the growth and proliferation of stem cells, and is not easy to change the morphology. In 20% serum, cell growth is fast, but easy to change the morphology.
3. Flow cytometry analysis: CD29 (+), CD44 (+), CD34 (-), CD45 (-), consistent with the characteristics of mesenchymal stem cell surface markers. RT-PCR analysis: mesenchymal stem cells do not express ALB, AFP and CK19.
4. After 8 passages of mesenchymal stem cells in vitro, the proliferation ability of mesenchymal stem cells was significantly weakened, and the cells became larger and irregular. At the 10th passage, the cells could not continue to pass on and died. After resuscitation, the mesenchymal stem cells would undergo a plateau period of 1 week before they could gradually recover to their pre-freezing state.
5. The 1st induction group (co-cultured with L02), the 2nd induction group (group A), the 3rd induction group (co-cultured with LO2 + group A) were induced for 6w. There were no morphological changes and no expression of ALB, AFP and CK19 mRNA in the control group.
6. The expression of ALB, AFP and CK19 mRNA was not detected in the 4th induction group (group B).
7. In group 5 (group C), AFP mRNA was detected by RT-PCR after 4 weeks of induction. AFP mRNA, ALB mRNA and CK19 mRNA were detected after 2 weeks of induction.
Conclusion:
Umbilical cord blood MSCs adhere to the wall later than bone marrow MSCs, so it is difficult to culture in vitro. In order to achieve in vitro amplification, high density inoculation is needed. A high serum concentration of 10-15% is appropriate. But serum concentration should not be too high, too high serum concentration (>20%) can promote fine. Cell differentiation and proliferation ability were significantly reduced.
2. MSCs cultured in vitro have limited ability of passage and good viability of P4-P8 generation. P10 generation cells die, so it is impossible to proliferate MSCs indefinitely in vitro. After cryopreservation, MSCs grow slowly after resuscitation. It needs a plateau period of one week to restore the state before cryopreservation.
3. It is very difficult to induce umbilical cord blood MSCs to differentiate into hepatocytes in vitro. In this experiment, polygonal hepatocyte-like cells were obtained in 5 induction groups only by stage 5 induction method. AFP mRNA, ALB mRNA and CK19 mRNA were detected by RT-PCR in these cells.
4. In the process of induction, the cell adherence ability is poor, the cell viability is weak, and the proliferation is difficult. How to achieve in vitro expansion of the induced cells, so as to be able to apply in clinical is the next step to solve the problem.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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