诱导新生狨猴皮肤成纤维细胞来源的多潜能干细胞的研究

发布时间：2018-04-17 14:36

本文选题：多潜能干细胞 + 转录因子　；参考：《西北农林科技大学》2010年博士论文

【摘要】：诱导性多潜能干细胞(induced pluripotent stem cells,iPSCs )的研究尚处于初级阶段,仍存在许多技术问题,其中之一是细胞重编程效率太低。目前,很多提高体细胞重编程效率的方法都集中在细胞诱导之后的细胞重编程过程,或是不同的诱导方法,而对提高两个或多个病毒共感染细胞的效率方面尚未进行过系统全面的研究报道。本研究首先用高浓度聚凝胺浓缩病毒结合低速离心的方法感染与狨猴同源性相近的人成纤维细胞(PHF),优化逆转录病毒感染细胞的方案,并且应用小鼠(3T3)成纤维细胞进一步检测优化的感染细胞方法的可行性;其次,用优化的逆转录病毒感染细胞的方法将4种转录因子(hOct4,hSox2,hKlf4和hc-Myc)基因导入新生狨猴皮肤成纤维细胞,首次将其诱导成为miPSCs;同时建立了miPSCs的无饲养层培养体系,并且通过培养液中添加Rho-激酶抑制剂Y27632来促进miPSCs的单细胞的存活和增殖。 1、分别以人成纤维细胞和3T3细胞为靶细胞,双嗜性包装细胞系Plat-A生产的表达绿色荧光蛋白pLEGFP-N1和表达红色荧光蛋白pMXs-dsRed2基因病毒按体积比为1:1(相同病毒滴度混合)的比例混合,比较未浓缩病毒的普通方法,优化的离心感染细胞的方法(spinoculation),超速离心浓缩病毒(10,000 g),高浓度聚凝胺(Polybrene)浓缩病毒( Flocculation )以及浓缩病毒结合离心感染细胞的方法(spinoculation/Flocculation)对共转染效率的影响,结果表明320μg/ml高浓度聚凝胺浓缩病毒(Flocculation)同低速离心感染细胞(spinoculation)相结合的方法将共转染率由普通方法的4.0±0.1%(PHF)和3.4±0.2%(3T3)分别提高到了53.0±0.6%(PHF)和35.0±0.4%(3T3)。 2、用优化的逆转录病毒共转染的方法将逆转录病毒pMXs-hOct4,hSox2,hKlf4,hc-Myc 4种转录因子基因导入新生狨猴皮肤成纤维细胞(MF),用1mM丙戊酸的培养液处理12天。感染4×10~5 MF,获得大约100个iPSCs样克隆,克隆分离培养于MEF饲养层上。其中30个克隆能够扩大培养,并冷冻保存。从中挑选8个克隆作为进一步的生物学特性研究,具有典型胚胎干细胞的形态特征,碱性磷酸酶染色呈阳性;qPCR结果表明表达很高的Nanog,Oct4和Sox2 mRNAs而相应的载体基因保持沉默;具有正常的染色体核型;免疫细胞化学表明表达胚胎干细胞特异性标志Oct4以及阶段特异性胚胎表面抗原SSEA-4,肿瘤排斥抗原TRA-1-81;于严重联合性免疫缺陷小鼠(RAG2-/-,c-/-)体内形成具有内、中、外三个胚层不同组织结构的畸胎瘤。因此,所得miPSCs是完全重编程细胞。 3、以MEF,STO分别制备条件培养液,比较了不同条件培养液对miPSCs的影响;同时,本试验以MEF为条件培养液比较了不同浓度血清(FBS)(10 %,15 %和20 %),不同浓度的条件培养液(35 %和17.5 %)以及不同浓度bFGF(20 ng/ml,100 ng/ml和150 ng/ml)对miPSCs的影响。通过qPCR的方法研究Oct4,Sox2,nanog表达的影响,于不同浓度FBS、条件培养液和bFGF中基因表达水平没有差异,并且同MEF为饲养层培养的miPSCs的基因表达水平没有差异;无饲养层条件下miPSCs碱性磷酸酶呈阳性;免疫细胞化学表明表达胚胎干细胞特异性标志Oct4以及表面抗原TRA-1-81和SSEA-3,SSEA-4;具有正常的核型;体外能够分化为神经元,严重联合性免疫缺陷小鼠(RAG2-/-,c-/-)体内形成具有三个胚层不同组织结构的畸胎瘤,说明无饲养层培养体系适用于miPSCs。 4、用Accutase将miPSCs消化为单个细胞,通过将无饲养层培养液中添加10μmol/L Y27632,培养低密度miPSCs检验Y27632的影响,结果表明Y27632能够促进miPSCs存活,其克隆效率由5.0±0.4 %提高到33.0±6.7 % (P0.01);提高冷冻复苏细胞的克隆效率到36.0±6.7 %(P0.01);BrdU免疫荧光染色表明,Y27632能够促进miPSCs的增殖,BrdU阳性细胞数由27.0±2.1 %提高到了34.0±1.8 %(P0.01);qPCR、免疫细胞化学表明,仍表达Nanog,Oct4和Sox2;具有正常的核型;能够体外分化为神经元,严重联合性免疫缺陷小鼠体内形成具有三个胚层组织的畸胎瘤,因此,10μmol/L Y27632培养条件下的miPSCs仍具有多能性。
[Abstract]:Induced pluripotent stem cells (induced pluripotent stem cells, iPSCs) the research is still in the initial stage, there are still many technical problems, one of which is the cell reprogramming efficiency is too low. At present, many cell reprogramming of somatic cell reprogramming improve the efficiency of the method are concentrated in cells after induced, or induced different methods, and to improve the two or more virus co infection efficiency of cells has not yet been systematic research reports comprehensively.
In this study, with a high concentration of Polybrene concentrated virus combined with low speed centrifugal method infection and marmoset homologous Their natures are much the same. human fibroblast (PHF) cells, optimization of retroviral infection, and application of mice (3T3) infected cells and fibroblasts feasibility method further optimized detection; secondly, using the method of optimization retrovirus infected cells will be 4 transcription factors (hOct4, hSox2, hKlf4 and hc-Myc) gene into neonatal marmoset skin fibroblasts, the first to be induced miPSCs; while establishing a feeder free culture system of miPSCs, and through the medium adding Rho- kinase inhibitor Y27632 to promote miPSCs single cell survival and proliferation.
1, with human fibroblasts and 3T3 cells as target cells, eosinophils double expression of green fluorescent protein pLEGFP-N1 packaging cell line Plat-A production and expression of red fluorescent protein gene of pMXs-dsRed2 virus according to the volume ratio of 1:1 (the same virus titer mixed) mixture was compared with common methods for virus concentration, optimization of centrifugal method the infected cells (spinoculation), ultracentrifugation virus concentration (10000 g), high concentration of Polybrene (Polybrene) virus concentration (Flocculation) method of centrifugal infected cells and concentrated virus binding (spinoculation/Flocculation) effect on CO transfection efficiency. The results show that 320 g/ml high concentration of Polybrene (Flocculation) with concentrated virus low speed centrifugation of infected cells (spinoculation) method combining co transfection rate by way of 4 + 0.1% and 3.4 + 0.2% (PHF) (3T3) were increased to 53 + 0.6% (PHF) and 35 + 0.4% (3T3).
2, using the method of optimization of retroviral co transfected with the retroviral pMXs-hOct4, hSox2, hKlf4, hc-Myc 4 transcription factor gene into neonatal marmoset skin fibroblasts (MF), 1mM valproic acid culture medium for 12 days. The infection of 4 * 10~5 MF, about 100 iPSCs like cloning, cloning cultured on MEF feeder layer. Among the 30 clones can expand cultivation and cryopreservation. 8 clones from the biological characteristics as the research further, with the morphological characteristics of embryonic stem cells, alkaline phosphatase staining was positive; qPCR showed high expression of Nanog, Oct4 and Sox2 mRNAs and the corresponding gene carrier keep silent; with normal karyotype; Immunocytochemistry showed that the expression of embryonic stem cell specific markers Oct4 and stage specific embryonic antigen SSEA-4, tumor rejection antigen TRA-1-81 in severe combined; In RAG2-/- (c-/-), there are three teratoma with inner, outer and outer miPSCs.
In 3, MEF, STO were prepared by conditioned medium, different conditioned medium effect on miPSCs; at the same time, the experiment with MEF conditioned medium compared with the different concentration of serum (FBS) (10%, 15% and 20%), different concentrations of conditioned medium (35% and 17.5%) and different the concentration of bFGF (20 ng/ml, 100 ng/ml and 150 ng/ml) effect on miPSCs. Through the study of Oct4, qPCR Sox2, expression of Nanog, FBS in different concentration, culture conditions did not differ between the gene expression level of liquid and bFGF, and with MEF as feeder layer culture of miPSCs gene expression level was no difference; without feeder layer miPSCs under the condition of alkaline phosphatase was positive; Immunocytochemistry showed that the expression of embryonic stem cell specific markers Oct4 and surface antigen TRA-1-81 and SSEA-3, SSEA-4; with normal karyotype; in vitro can differentiate into neurons, severe combined immunodeficiency mice (R AG2-/-, c-/-) formation of teratoma with three different tissue structures in the body, indicating that no feeder culture system is suitable for miPSCs.
4, Accutase miPSCs will be digested into single cells, the feeder free medium with 10 mol/L Y27632 training, training effect of low density miPSCs Y27632 test, the results show that Y27632 can promote miPSCs survival, the cloning efficiency is increased from 5 + 0.4% to 33 + 6.7% (P0.01); to improve the efficiency of cloning of cryopreserved cells to 36 + 6.7% (P0.01); BrdU immunofluorescence staining showed that Y27632 can promote the proliferation of miPSCs, the number of BrdU positive cells from 27 + 2.1% to 34 + 1.8% (P0.01); qPCR, immunocytochemistry showed that the expression of Oct4 and Sox2 is Nanog; with normal karyotype can differentiate into in vitro; the formation of neurons, severe combined immunodeficient mice with three germ layers of teratoma, therefore, 10 mol/L Y27632 culture condition miPSCs still have pluripotency.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

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