本文選題：造血干細(xì)胞 + 間充質(zhì)干細(xì)胞��； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年碩士論文

【摘要】：引言研究表明自懷孕第6周至妊娠結(jié)束,胎盤中均含有HSC(Hematopoietic stem cell,HSC),且具有細(xì)胞數(shù)量大,獲取便捷,使用過(guò)程中不存在倫理問(wèn)題等優(yōu)點(diǎn)。目前針對(duì)臍血(Umbilical cord blood,UCB)來(lái)源造血干/祖細(xì)胞(Hematopoietic stem and progenitor cell,HSPC)的體外擴(kuò)增臨床研究已經(jīng)取得成功,采用擴(kuò)增后UCB來(lái)源HSPC進(jìn)行移植可以有效解決HSC數(shù)量不足造成的造血恢復(fù)延遲等問(wèn)題,數(shù)據(jù)表明移植后干細(xì)胞較未擴(kuò)增UCB來(lái)源HSPC具有相同植入率。胎盤來(lái)源HSC與UCB來(lái)源HSC同屬于圍產(chǎn)期干細(xì)胞,研究表明其較UCB來(lái)源HSC更為原始,目前國(guó)內(nèi)外尚無(wú)針對(duì)胎盤來(lái)源HSC體外擴(kuò)增的文獻(xiàn)報(bào)道。目的本研究旨在通過(guò)比較機(jī)械處理聯(lián)合化學(xué)酶消化法及AMD3100循環(huán)灌注法提取人胎盤來(lái)源HSC的效果,構(gòu)建適用于未來(lái)臨床應(yīng)用的胎盤來(lái)源HSC分離提取方法,得到來(lái)源清楚組份確定的HSC;基于模擬造血微環(huán)境的體外擴(kuò)增理論,探討UCB中CD34+細(xì)胞在間充質(zhì)干細(xì)胞(Mesenchymal stem cell,MSC)共培養(yǎng)體系、UM171聯(lián)合細(xì)胞因子擴(kuò)增體系以及UM171聯(lián)合細(xì)胞因子與MSC共培養(yǎng)體系三種不同擴(kuò)增體系下體外擴(kuò)增效果;依據(jù)優(yōu)化的胎盤來(lái)源HSC提取方法,對(duì)胎盤來(lái)源HSC進(jìn)行分離提取,并基于模擬造血微環(huán)境理論構(gòu)建的擴(kuò)增體系對(duì)提取得到的CD34+細(xì)胞行體外擴(kuò)增,評(píng)價(jià)擴(kuò)增效果及擴(kuò)增后HSPC功能,為拓展臨床HSC來(lái)源做出嘗試。方法1、健康足月順產(chǎn)胎盤依照處理方法不同分為兩組進(jìn)行胎盤來(lái)源HSPC體外提取:A組(機(jī)械處理聯(lián)合化學(xué)酶消化組)、B組(AMD3100循環(huán)灌注組),每組處理胎盤5個(gè)。A組將胎盤經(jīng)預(yù)處理后解剖成小組織塊,采用磷酸鹽緩沖液(Phosphate buffered saline,PBS)清洗后收集清洗液(命名為A1),采用膠原酶、透明質(zhì)酸酶、DNA酶對(duì)清洗后的胎盤組織塊進(jìn)行消化,并置于100目及200目濾網(wǎng)研磨,收集消化液(命名為A2)。B組采用智能蠕動(dòng)泵連接臍動(dòng)靜脈,采用生理鹽水(Normal saline,NS)NS及含AMD3100的NS分步對(duì)胎盤脈管系統(tǒng)行循環(huán)灌注,分別收集兩次灌洗液(命名為B1及B2)。比較兩種方法收集得到各類細(xì)胞總數(shù),并分別測(cè)定兩種方法不同階段收集得到細(xì)胞數(shù)量及其流式表型情況;2、采集到的UCB進(jìn)行磁珠分選后得到純凈的CD34+細(xì)胞,臍帶剪至小塊后采用含有0.2%Ⅱ型膠原酶的DMEM/F12培養(yǎng)基進(jìn)行消化后,進(jìn)行貼壁傳代培養(yǎng)。UCB來(lái)源CD34+細(xì)胞及臍帶來(lái)源MSC分為以下四組進(jìn)行體外擴(kuò)增培養(yǎng)10天:A組(對(duì)照組)、B組(UM171培養(yǎng)組)、C組(MSC共培養(yǎng)組)、D組(UM171聯(lián)合MSC共培養(yǎng)組)。四組細(xì)胞均采用StemSpan培養(yǎng)基,并添加100 ng/ml干細(xì)胞因子(Stem cell factor,SCF),100 ng/ml FMS樣酪氨酸激酶3配體(FMS-like trysine kinase 3ligand,FLT3),50 ng/ml促血小板生成素(thrombopoietin,TPO),CD34+按照1×105/孔密度接種于六孔板中,其中B組在培養(yǎng)基中額外添加100 ng/ml UM171,C組采用分離得到的第三代臍血同源臍帶MSC作為基質(zhì)細(xì)胞,待貼壁90%融合采用60Co照射,劑量為10Gy,D組在C組實(shí)驗(yàn)條件基礎(chǔ)上同時(shí)添加100 ng/ml UM171,細(xì)胞培養(yǎng)10天后比較不同組別間細(xì)胞擴(kuò)增倍數(shù)及流式表型和集落培養(yǎng)情況;3、分別將AMD3100循環(huán)灌注法收集的NS灌洗液及AMD3100灌洗液經(jīng)免疫磁珠純化,采用UM171聯(lián)合MSC共培養(yǎng)方法分別對(duì)純化后的胎盤來(lái)源CD34+細(xì)胞進(jìn)行體外擴(kuò)增培養(yǎng),驗(yàn)證其擴(kuò)增效果及流式表型和集落培養(yǎng)情況。結(jié)果1、A組與B組收集得到的TNC分別為(45.19±9.41)×107和(42.69±11.19)×107,二者無(wú)統(tǒng)計(jì)學(xué)差異;收集得到的CD34+細(xì)胞數(shù)(12.98±3.62)×107和(1.87±0.87)×107,二者比較,p=0.00,具有顯著統(tǒng)計(jì)學(xué)差異;收集得到的CD133+細(xì)胞數(shù)分別為(1.16±0.61)×107和(1.15±0.63)×107,二者無(wú)統(tǒng)計(jì)學(xué)差異;A組收獲的細(xì)胞液A1中含有TNC為(6.76±2.13)×107,其中CD34+CD38-比例為(1.14±0.56)%。收獲的細(xì)胞液A2中含有TNC為(38.44±7.33)×107,其中CD34+CD38-比例為(30.31±10.75)%;B組收獲的細(xì)胞液B1中含有TNC為(15.11±6.03)×107,其中CD34+CD38-比例為(0.72±0.34)%。收獲的細(xì)胞液B2中含有TNC為(27.58±11.14)×107,其中CD34+CD38-比例為(5.56±1.78)%;2、分離得到的臍帶MSC傳代培養(yǎng)后細(xì)胞狀態(tài)良好,流式檢測(cè)發(fā)現(xiàn)其高表達(dá)CD105,CD73,CD90,不表達(dá)CD14,CD34,CD19,CD45,HLA-DR,SSEA-4含量約在1%,經(jīng)過(guò)誘導(dǎo)培養(yǎng)后可以分化成骨細(xì)胞、脂肪細(xì)胞、軟骨細(xì)胞。CD34+細(xì)胞在不同條件下體外培養(yǎng)10天后發(fā)現(xiàn),對(duì)照組細(xì)胞總數(shù)擴(kuò)增3.7倍,CD34+細(xì)胞擴(kuò)增3.5倍,組別間無(wú)明顯差異。UM171培養(yǎng)組總有核細(xì)胞數(shù)擴(kuò)增14倍,CD34+細(xì)胞擴(kuò)增13.5倍;MSCs共培養(yǎng)組總有核細(xì)胞數(shù)擴(kuò)增11倍,CD34+細(xì)胞擴(kuò)增10倍;聯(lián)合培養(yǎng)組總有核細(xì)胞數(shù)擴(kuò)增達(dá)22倍,CD34+細(xì)胞擴(kuò)增21倍。UM171聯(lián)合MSCs組得到CD34+CD38-細(xì)胞比例為91.49±2.67%,較MSCs及UM171組(分別為(78.11±2.34)%,(87.66±1.48)%)均在顯著差異,含UM171培養(yǎng)體系CD133細(xì)胞比例較單獨(dú)MSCs培養(yǎng)組存在差異,擴(kuò)增后細(xì)胞集落培養(yǎng)14天后,各系集落形成良好,UM171擴(kuò)增組細(xì)胞較MSCs擴(kuò)增組在紅系及粒系形成能力方面存在優(yōu)勢(shì)。3、胎盤NS灌洗液中CD34+細(xì)胞經(jīng)10d培養(yǎng)后細(xì)胞總數(shù)擴(kuò)增17倍,CD34+細(xì)胞擴(kuò)增14倍;胎盤AMD3100灌洗液中CD34+細(xì)胞經(jīng)10天培養(yǎng)后幾乎全部死亡。臍血對(duì)照組與胎盤NS灌洗組擴(kuò)增后細(xì)胞在TNC、CD34+細(xì)胞總數(shù)、CD133+細(xì)胞總數(shù)均存在統(tǒng)計(jì)學(xué)差異,其中胎盤NS灌洗組CD34+CD38-比例為(82.70±6.89)%;擴(kuò)增后臍血來(lái)源及胎盤NS灌洗液中CD34+各譜系集落形成能力良好,胎盤NS灌洗液中CD34+細(xì)胞在粒系形成能力方面較未擴(kuò)增臍血CD34+細(xì)胞存在差異。結(jié)論1、機(jī)械處理聯(lián)合化學(xué)酶消化法以及AMD3100循環(huán)灌注法均可以對(duì)胎盤來(lái)源CD34+細(xì)胞進(jìn)行有效收集,其中AMD3100循環(huán)灌注法在保證細(xì)胞收集數(shù)量的同時(shí)可以有效降低細(xì)菌污染風(fēng)險(xiǎn),并可降低母體組織對(duì)胎盤來(lái)源HSC的影響,收集得到的CD34+細(xì)胞經(jīng)免疫磁珠分選后純度較高,較機(jī)械處理聯(lián)合化學(xué)酶消化法在收集胎盤來(lái)源HSC上具有更大的優(yōu)勢(shì);2、臍帶血間充質(zhì)干細(xì)胞作為細(xì)胞滋養(yǎng)層可提高CD34+細(xì)胞體外擴(kuò)增效果,UM171在擴(kuò)增過(guò)程中可較好的保持細(xì)胞干性,二者聯(lián)合應(yīng)用擴(kuò)增效果最佳,建立的臍帶間充質(zhì)干細(xì)胞聯(lián)合UM171對(duì)臍血源CD34+細(xì)胞的擴(kuò)增方法可用于CD34+細(xì)胞體外擴(kuò)增培養(yǎng)。3、經(jīng)AMD3100循環(huán)灌注法收集的NS灌洗液及AMD3100灌洗液中CD34+細(xì)胞在臍帶MSC聯(lián)合細(xì)胞因子及UM171培養(yǎng)體系中擴(kuò)增效果存在明顯差異,其中NS灌洗液中CD34+細(xì)胞擴(kuò)增效果可達(dá)17倍,其中較為原始的CD34+CD38—細(xì)胞比例達(dá)82%,擴(kuò)增后細(xì)胞集落培養(yǎng)實(shí)驗(yàn)表明其具有與臍血來(lái)源擴(kuò)增后CD34+細(xì)胞同樣的譜系重建能力。AMD3100灌洗液中CD34+細(xì)胞在該體系中不能進(jìn)行有效擴(kuò)增,其細(xì)胞來(lái)源及功能尚需進(jìn)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.

【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R329.2

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