胎盘来源造血干细胞分离提取和体外扩增研究

发布时间：2018-05-15 02:05

本文选题：造血干细胞 + 间充质干细胞　；参考：《中国人民解放军军事医学科学院》2017年硕士论文

【摘要】：引言研究表明自怀孕第6周至妊娠结束,胎盘中均含有HSC(Hematopoietic stem cell,HSC),且具有细胞数量大,获取便捷,使用过程中不存在伦理问题等优点。目前针对脐血(Umbilical cord blood,UCB)来源造血干/祖细胞(Hematopoietic stem and progenitor cell,HSPC)的体外扩增临床研究已经取得成功,采用扩增后UCB来源HSPC进行移植可以有效解决HSC数量不足造成的造血恢复延迟等问题,数据表明移植后干细胞较未扩增UCB来源HSPC具有相同植入率。胎盘来源HSC与UCB来源HSC同属于围产期干细胞,研究表明其较UCB来源HSC更为原始,目前国内外尚无针对胎盘来源HSC体外扩增的文献报道。目的本研究旨在通过比较机械处理联合化学酶消化法及AMD3100循环灌注法提取人胎盘来源HSC的效果,构建适用于未来临床应用的胎盘来源HSC分离提取方法,得到来源清楚组份确定的HSC;基于模拟造血微环境的体外扩增理论,探讨UCB中CD34+细胞在间充质干细胞(Mesenchymal stem cell,MSC)共培养体系、UM171联合细胞因子扩增体系以及UM171联合细胞因子与MSC共培养体系三种不同扩增体系下体外扩增效果;依据优化的胎盘来源HSC提取方法,对胎盘来源HSC进行分离提取,并基于模拟造血微环境理论构建的扩增体系对提取得到的CD34+细胞行体外扩增,评价扩增效果及扩增后HSPC功能,为拓展临床HSC来源做出尝试。方法1、健康足月顺产胎盘依照处理方法不同分为两组进行胎盘来源HSPC体外提取:A组(机械处理联合化学酶消化组)、B组(AMD3100循环灌注组),每组处理胎盘5个。A组将胎盘经预处理后解剖成小组织块,采用磷酸盐缓冲液(Phosphate buffered saline,PBS)清洗后收集清洗液(命名为A1),采用胶原酶、透明质酸酶、DNA酶对清洗后的胎盘组织块进行消化,并置于100目及200目滤网研磨,收集消化液(命名为A2)。B组采用智能蠕动泵连接脐动静脉,采用生理盐水(Normal saline,NS)NS及含AMD3100的NS分步对胎盘脉管系统行循环灌注,分别收集两次灌洗液(命名为B1及B2)。比较两种方法收集得到各类细胞总数,并分别测定两种方法不同阶段收集得到细胞数量及其流式表型情况;2、采集到的UCB进行磁珠分选后得到纯净的CD34+细胞,脐带剪至小块后采用含有0.2%Ⅱ型胶原酶的DMEM/F12培养基进行消化后,进行贴壁传代培养。UCB来源CD34+细胞及脐带来源MSC分为以下四组进行体外扩增培养10天:A组(对照组)、B组(UM171培养组)、C组(MSC共培养组)、D组(UM171联合MSC共培养组)。四组细胞均采用StemSpan培养基,并添加100 ng/ml干细胞因子(Stem cell factor,SCF),100 ng/ml FMS样酪氨酸激酶3配体(FMS-like trysine kinase 3ligand,FLT3),50 ng/ml促血小板生成素(thrombopoietin,TPO),CD34+按照1×105/孔密度接种于六孔板中,其中B组在培养基中额外添加100 ng/ml UM171,C组采用分离得到的第三代脐血同源脐带MSC作为基质细胞,待贴壁90%融合采用60Co照射,剂量为10Gy,D组在C组实验条件基础上同时添加100 ng/ml UM171,细胞培养10天后比较不同组别间细胞扩增倍数及流式表型和集落培养情况;3、分别将AMD3100循环灌注法收集的NS灌洗液及AMD3100灌洗液经免疫磁珠纯化,采用UM171联合MSC共培养方法分别对纯化后的胎盘来源CD34+细胞进行体外扩增培养,验证其扩增效果及流式表型和集落培养情况。结果1、A组与B组收集得到的TNC分别为(45.19±9.41)×107和(42.69±11.19)×107,二者无统计学差异;收集得到的CD34+细胞数(12.98±3.62)×107和(1.87±0.87)×107,二者比较,p=0.00,具有显著统计学差异;收集得到的CD133+细胞数分别为(1.16±0.61)×107和(1.15±0.63)×107,二者无统计学差异;A组收获的细胞液A1中含有TNC为(6.76±2.13)×107,其中CD34+CD38-比例为(1.14±0.56)%。收获的细胞液A2中含有TNC为(38.44±7.33)×107,其中CD34+CD38-比例为(30.31±10.75)%;B组收获的细胞液B1中含有TNC为(15.11±6.03)×107,其中CD34+CD38-比例为(0.72±0.34)%。收获的细胞液B2中含有TNC为(27.58±11.14)×107,其中CD34+CD38-比例为(5.56±1.78)%;2、分离得到的脐带MSC传代培养后细胞状态良好,流式检测发现其高表达CD105,CD73,CD90,不表达CD14,CD34,CD19,CD45,HLA-DR,SSEA-4含量约在1%,经过诱导培养后可以分化成骨细胞、脂肪细胞、软骨细胞。CD34+细胞在不同条件下体外培养10天后发现,对照组细胞总数扩增3.7倍,CD34+细胞扩增3.5倍,组别间无明显差异。UM171培养组总有核细胞数扩增14倍,CD34+细胞扩增13.5倍;MSCs共培养组总有核细胞数扩增11倍,CD34+细胞扩增10倍;联合培养组总有核细胞数扩增达22倍,CD34+细胞扩增21倍。UM171联合MSCs组得到CD34+CD38-细胞比例为91.49±2.67%,较MSCs及UM171组(分别为(78.11±2.34)%,(87.66±1.48)%)均在显著差异,含UM171培养体系CD133细胞比例较单独MSCs培养组存在差异,扩增后细胞集落培养14天后,各系集落形成良好,UM171扩增组细胞较MSCs扩增组在红系及粒系形成能力方面存在优势。3、胎盘NS灌洗液中CD34+细胞经10d培养后细胞总数扩增17倍,CD34+细胞扩增14倍;胎盘AMD3100灌洗液中CD34+细胞经10天培养后几乎全部死亡。脐血对照组与胎盘NS灌洗组扩增后细胞在TNC、CD34+细胞总数、CD133+细胞总数均存在统计学差异,其中胎盘NS灌洗组CD34+CD38-比例为(82.70±6.89)%;扩增后脐血来源及胎盘NS灌洗液中CD34+各谱系集落形成能力良好,胎盘NS灌洗液中CD34+细胞在粒系形成能力方面较未扩增脐血CD34+细胞存在差异。结论1、机械处理联合化学酶消化法以及AMD3100循环灌注法均可以对胎盘来源CD34+细胞进行有效收集,其中AMD3100循环灌注法在保证细胞收集数量的同时可以有效降低细菌污染风险,并可降低母体组织对胎盘来源HSC的影响,收集得到的CD34+细胞经免疫磁珠分选后纯度较高,较机械处理联合化学酶消化法在收集胎盘来源HSC上具有更大的优势;2、脐带血间充质干细胞作为细胞滋养层可提高CD34+细胞体外扩增效果,UM171在扩增过程中可较好的保持细胞干性,二者联合应用扩增效果最佳,建立的脐带间充质干细胞联合UM171对脐血源CD34+细胞的扩增方法可用于CD34+细胞体外扩增培养。3、经AMD3100循环灌注法收集的NS灌洗液及AMD3100灌洗液中CD34+细胞在脐带MSC联合细胞因子及UM171培养体系中扩增效果存在明显差异,其中NS灌洗液中CD34+细胞扩增效果可达17倍,其中较为原始的CD34+CD38—细胞比例达82%,扩增后细胞集落培养实验表明其具有与脐血来源扩增后CD34+细胞同样的谱系重建能力。AMD3100灌洗液中CD34+细胞在该体系中不能进行有效扩增,其细胞来源及功能尚需进一步研究。
[Abstract]:The introduction shows that from sixth weeks to the end of pregnancy, the placenta contains HSC (Hematopoietic stem cell, HSC), and has the advantages of large number of cells, convenient access and no ethical problems in the process of use. At present, the origin of hematopoietic stem / progenitor cells (Hematopoietic stem and) is based on the umbilical cord blood (Umbilical cord blood, UCB). The clinical study of in vitro amplification has been successful. The use of amplified UCB source HSPC for transplantation can effectively solve the problem of the delay of hematopoiesis caused by the insufficient number of HSC. The data show that the transplanted stem cells have the same implantation rate compared with the non amplification UCB source HSPC. The placental source HSC and UCB source HSC belong to perinatal stem cells, which belong to the perinatal stem cells. It shows that it is more primitive than UCB source HSC, and there is no literature report on the expansion of HSC in vitro at home and abroad. The purpose of this study is to construct the extraction of HSC from human placenta by comparison of mechanical processing combined with chemical enzyme digestion and AMD3100 circulation perfusion method, and to construct the separation and extraction of HSC from placental sources for future clinical application. Methods the HSC was determined by the source clear component, and based on the in vitro amplification theory of simulated hematopoietic microenvironment, the co culture system of CD34+ cells (Mesenchymal stem cell, MSC) in UCB, the joint cytokine amplification system of UM171 and the three different amplification systems of the co culture system of UM171 combined with MSC were discussed. According to the optimized placental source HSC extraction method, the placental source HSC was isolated and extracted, and the extracted CD34+ cells were amplified in vitro based on the analogue hematopoietic microenvironment theory. The effect of amplification and the function of HSPC after amplification were evaluated. Method 1, healthy full term delivery was made. Placentas were divided into two groups according to different treatment methods: placenta source HSPC extracorporeal extraction: A group (mechanical treatment combined with chemical enzyme digestion group), group B (AMD3100 cycle perfusion group), each group of placenta treated groups to dissection the placenta after preprocessing and dissection into group fabric, using phosphate buffer solution (Phosphate buffered saline, PBS) after cleaning to collect cleaning. The liquid (named as A1) was digested with collagenase, hyaluronidase and DNA enzyme to the cleaned placental tissue blocks, and was lapping in 100 mesh and 200 mesh filters. The collection of digestive juice (named A2).B was connected to the umbilical vein by an intelligent peristaltic pump, and the Normal saline, NS NS and NS with AMD3100 were used to follow the placental vascular system. Two times of lavage were collected (named B1 and B2). The total number of cells was collected by two methods. The number of cells and the flow phenotype of the cells were collected at different stages of two methods. 2, after the collection of UCB, the pure CD34+ cells were obtained after the magnetic beads were selected. The umbilical cord was cut to a small block, and 0.2% II was used. After digesting the DMEM/F12 culture medium of type collagenase, the.UCB source CD34+ cells and the umbilical cord source were divided into four groups for 10 days in vitro: A group (control group), B group (UM171 culture group), C group (MSC co culture group), D group (UM171 coupling MSC co culture group). The four groups of cells all used the culture medium and added 100 ng/ml stem cell factor (Stem cell factor, SCF), 100 ng/ml FMS like tyrosine kinase 3 ligands (FMS-like trysine kinase 3ligand, FLT3), 50 thrombopoietin (thrombopoietin) were inoculated in the six pore plate according to the density of 1 x aperture. The third generation umbilical cord blood umbilical cord MSC was used as the matrix cell, and the adherent wall 90% was irradiated with 60Co, the dose was 10Gy, and the D group added 100 ng/ml UM171 on the basis of the experimental conditions of the C group. After 10 days of cell culture, the cell amplification multiple, the flow phenotype and the colony culture condition were compared between the different groups. 3, the AMD3100 circulation perfusion method was collected respectively. NS lavage fluid and AMD3100 lavage liquid were purified by immunomagnetic beads. The purified CD34+ cells were amplified and cultured in vitro by UM171 combined with MSC co culture method. The amplification effect, flow phenotype and colony culture of the purified placenta derived CD34+ cells were verified. Results 1, the TNC of A group and B group was (45.19 + 9.41) * 107 and (42.69 + 11.19) x 10, respectively. 7, there were no statistical differences, the number of CD34+ cells collected was (12.98 + 3.62) x 107 and (1.87 + 0.87) x 107, and the two were compared with p=0.00, with significant statistical differences. The number of CD133+ cells collected was (1.16 + 0.61) x 107 and (1.15 + 0.63), respectively, and there was no statistical difference, and TNC was found in A1 of group A. 07, the proportion of CD34+CD38- was (1.14 + 0.56)%. The A2 in the harvested cell liquid contained TNC (38.44 + 7.33) x 107, and the proportion of CD34+CD38- was (30.31 + 10.75)%, and the B1 in the B group contained TNC was (15.11 + 6.03) * 107, and CD34+CD38- ratio was (0.72 + 0.34)%. The B2 contained TNC for the harvested cell liquid, which was CD34. The proportion of +CD38- was (5.56 + 1.78)%; 2, the isolated umbilical cord MSC was cultured in good condition. Flow cytometry found that its high expression of CD105, CD73, CD90, did not express CD14, CD34, CD19, CD45, HLA-DR, SSEA-4 content was about 1%, and could be differentiated into osteoblasts, adipocytes and chondrocyte.CD34+ cells under different conditions after induction culture. After 10 days of culture, the total number of cells in the control group amplified 3.7 times, the CD34+ cells expanded 3.5 times, there was no significant difference between the group and the.UM171 culture group, the number of nuclear cells was 14 times, the CD34+ cell amplification was 13.5 times, the total number of nuclear cells in the MSCs co culture group was 11 times and the CD34+ cells expanded 10 times; the total number of nuclear cells in the combined culture group was 22 times and CD34+ thin. The proportion of CD34+CD38- cells in the group of 21 times.UM171 combined with MSCs was 91.49 + 2.67%, compared with MSCs and UM171 group (78.11 + 2.34)% and (87.66 + 1.48)% respectively), the proportion of CD133 cells in the UM171 culture system was different from that of the single MSCs culture group. After the amplification of cell colony for 14 days, the colony formation was good and UM171 amplification. The group cells were superior to the MSCs amplification group in the red and granulating capacity of.3. The total number of CD34+ cells in the placental NS lavage solution expanded 17 times after 10d culture and 14 times the amplification of CD34+ cells. The CD34+ cells in the placental AMD3100 lavage solution almost all died after 10 days. The cells in the umbilical blood control group and the placental NS lavage group were in TNC, C. The total number of D34+ cells and the total number of CD133+ cells were statistically different, of which the proportion of CD34+CD38- in the placental NS lavage group was (82.70 + 6.89)%, and the colony formation ability of CD34+ lineage in the umbilical cord blood and the placental NS lavage fluid was good, and the difference in the granulating capacity of CD34+ cells in the NS lavage fluid of the placenta was more than that of the non amplified cord blood CD34+ cells. Conclusion 1, mechanical treatment combined with chemical enzyme digestion and AMD3100 circulation perfusion method can effectively collect CD34+ cells from placenta, and AMD3100 circulation perfusion method can effectively reduce the risk of bacterial contamination while ensuring the number of cell collection, and can reduce the effect of maternal body fabric on the HSC of placenta, and collect CD34 The purity of + cells was higher after the separation of the immunomagnetic beads. The combined chemical enzyme digestion method was more advantageous than the mechanical treatment combined with chemical enzyme digestion. 2, the umbilical cord blood mesenchymal stem cells as the cell trophoblast could improve the amplification effect of CD34+ cells in vitro, and UM171 could keep the cell dry in the process of amplification, and the two were combined with amplification. The effect is best. The expansion method of umbilical cord mesenchymal stem cells combined with UM171 on umbilical cord blood CD34+ cells can be used for CD34+ cells to expand and culture.3 in vitro. The amplification effect of CD34+ cells in NS lavage solution collected by AMD3100 circulation perfusion method and AMD3100 lavage solution in the cord MSC combined cytokine and UM171 culture system in the umbilical cord is obviously different. The amplification effect of CD34+ cells in the NS lavage solution could reach 17 times, of which the proportion of the original CD34+CD38 cells was 82%. After the amplification, the cell colony culture experiment showed that it had the same lineage reconstruction ability of the CD34+ cells as the umbilical cord blood, and the CD34+ cells in the.AMD3100 lavage solution could not be amplified effectively in the system. The source and function need to be further studied.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2

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