本文選題：人臍血源基質(zhì)細(xì)胞 + 間隙連接蛋白43��； 參考：《第三軍醫(yī)大學(xué)》2017年博士論文

【摘要】：背景:造血干細(xì)胞移植是治療急性白血病的有效手段,自體造血干細(xì)胞移植具有適用范圍廣、費用較少、安全性高等優(yōu)點,但是移植后患者的復(fù)發(fā)率較高。微小殘留病(Minimal Residual Disease,MRD)清除不徹底是其根源。造血微環(huán)境對白血病的生長、支持、庇護(hù)是白血病殘留的重要因素。基質(zhì)細(xì)胞是造血微環(huán)境的主要成分,具有調(diào)控白血病細(xì)胞生長、浸潤,介導(dǎo)白血病的殘留及耐藥的作用。間隙連接細(xì)胞間通訊(gap junction intercellular communication,GJIC)是相鄰細(xì)胞間普遍存在的直接通訊方式。間隙連接蛋白43(connexin43,CX43)介導(dǎo)的GJIC廣泛存在于骨髓基質(zhì)細(xì)胞、基質(zhì)細(xì)胞與造血細(xì)胞之間,是骨髓基質(zhì)參與造血調(diào)控的必要條件。GJIC減弱或缺失在實體腫瘤的發(fā)生發(fā)展中起著重要作用。我們前期對急性白血病患者的骨髓基質(zhì)細(xì)胞進(jìn)行檢測發(fā)現(xiàn),其CX43表達(dá)明顯降低,GJIC功能也明顯減弱,對白血病的阻抑作用減弱;臨床研究發(fā)現(xiàn),自體造血干細(xì)胞移植后患者的GJIC功能長時間處于較低水平,導(dǎo)致骨髓微環(huán)境對白血病的阻抑作用減弱,可能是殘留白血病易復(fù)發(fā)的因素。人臍血源基質(zhì)細(xì)胞(human umbilical cord blood-derived stromal cells,hUCSCs)在體外對白血病細(xì)胞株具有抑制增殖及促進(jìn)凋亡的作用,體內(nèi)能歸巢至骨髓修復(fù)造血微環(huán)境。能否利用人臍血源基質(zhì)細(xì)胞這一基因載體和對白血病細(xì)胞具有阻抑作用的特性,將Cx43基因轉(zhuǎn)染新型人臍血源基質(zhì)細(xì)胞與自體造血干細(xì)胞聯(lián)合移植給預(yù)處理后的微小殘留病小鼠模型,起到重建移植后GJIC功能以凈化小鼠微小殘留病的作用,從而降低自體移植后白血病的復(fù)發(fā)是值得驗證的科學(xué)設(shè)想。本課題為國家自然科學(xué)基金項目(No.81070388)“CX43修飾人臍血源基質(zhì)細(xì)胞移植對自體造血干細(xì)胞移植(autologous hematopoietic stem cell transplantation,AHSCT)后殘留白血病凈化效應(yīng)及機(jī)制研究”的研究內(nèi)容。試圖從改造白血病造血微環(huán)境功能GJIC角度去影響白血病細(xì)胞的增殖、分化過程,從而達(dá)到去惡化的目的,為探尋白血病治療的新方法,積累理論和實驗依據(jù)奠定基礎(chǔ)。目的:觀察Cx43修飾的新型人臍血源基質(zhì)細(xì)胞移植在造血干細(xì)胞移植后的微小殘留白血病小鼠骨髓微環(huán)境的定植和對異常骨髓微環(huán)境的修復(fù)作用,通過修復(fù)和增強(qiáng)移植后小鼠骨髓基質(zhì)GJIC功能,改善骨髓微環(huán)境造血調(diào)控功能,實現(xiàn)對小鼠殘留白血病體內(nèi)凈化。方法:1.CX43重組腺病毒載體(Ad-Cx43-GFP)轉(zhuǎn)染人臍血源基質(zhì)細(xì)胞按本科室已經(jīng)建立的方法對健康產(chǎn)婦臍帶血進(jìn)行細(xì)胞分離并貼壁培養(yǎng),獲得人臍血源基質(zhì)細(xì)胞。按我科常規(guī)方法構(gòu)建Ad-Cx43-GFP重組腺病毒載體并轉(zhuǎn)染人臍血源基質(zhì)細(xì)胞。分別用免疫熒光法、PCR、Western-Blot、光脫色熒光恢復(fù)技術(shù)(fluorescence recovery after photo bleaching,FRAP)檢測轉(zhuǎn)染后Cx43表達(dá)。2.構(gòu)建人臍血源基質(zhì)細(xì)胞及L615細(xì)胞共培養(yǎng)體系,體外觀察CX43轉(zhuǎn)染前后人臍血源基質(zhì)細(xì)胞對L615白血病細(xì)胞的調(diào)節(jié)作用實驗分三組:1)陰性對照組:L615細(xì)胞單獨培養(yǎng);2)hUCSCs/L615組:人臍血源基質(zhì)細(xì)胞與L615細(xì)胞共培養(yǎng)組3)Ad-Cx43-hUCSCs/L615組:Ad-Cx43-GFP轉(zhuǎn)染的人臍血源基質(zhì)細(xì)胞與L615細(xì)胞共培養(yǎng)組CCK8法檢測共培養(yǎng)后L615細(xì)胞的生長曲線,對比各組間L615細(xì)胞增殖率;流式細(xì)胞術(shù)檢測共培養(yǎng)后L615細(xì)胞的凋亡率及細(xì)胞周期;Western-Blot檢測共培養(yǎng)后L615細(xì)胞caspase3、6、7表達(dá)情況。3.構(gòu)建L615白血病MRD小鼠模型,進(jìn)行人臍血源基質(zhì)細(xì)胞聯(lián)合自體造血干細(xì)胞移植,觀察轉(zhuǎn)染后人臍血源基質(zhì)細(xì)胞對自體造血干細(xì)胞移植小鼠殘留白血病的抑制效應(yīng)將L615細(xì)胞經(jīng)尾靜脈注入L615同系小鼠,3天后經(jīng)腹腔注射環(huán)磷酰胺200mg/kg,從而獲得L615白血病MRD小鼠模型。實驗分為兩組:1)BM組:骨髓造血干細(xì)胞移植組2)Ad-Cx43-hUCSCs+BM組:Ad-Cx43-GFP轉(zhuǎn)染的人臍血源基質(zhì)細(xì)胞聯(lián)合骨髓造血干細(xì)胞移植組以殘留白血病L615為受體鼠,以正常L615為供鼠,受鼠在第0天接受6.0 Gy 60Coγ射線TBI;照射后6 h,經(jīng)尾靜脈注射來自供鼠的骨髓造血干細(xì)胞細(xì)胞以及CM-Dil標(biāo)記的Cx43修飾的人臍血源基質(zhì)細(xì)胞,其中,BM組輸注骨髓細(xì)胞為2×106個/只、Ad-Cx43-hUCSCs+BM組輸注骨髓細(xì)胞及人臍血源基質(zhì)細(xì)胞各1×106個/只。對不同組小鼠移植后尾靜脈血行血常規(guī)檢查以觀察造血重建情況;對不同組小鼠移植后的骨髓進(jìn)行涂片及骨髓病理切片檢查;利用熒光標(biāo)記追蹤Ad-Cx43-hUCSCs移植后體內(nèi)分布情況;對移植后小鼠的骨髓基質(zhì)細(xì)胞進(jìn)行GJIC功能檢測,于移植后不同時間點分別取肝臟、脾臟、肺、腎臟進(jìn)行病理切片及HE染色;觀察各組小鼠移植后的生存率。結(jié)果:1.成功構(gòu)建Ad-Cx43-GFP重組腺病毒載體并轉(zhuǎn)染人臍血源基質(zhì)細(xì)胞:轉(zhuǎn)染后24h可見綠色熒光表達(dá),48h熒光表達(dá)達(dá)到峰值,轉(zhuǎn)染效率(89.30±3.12)%。免疫熒光、RT-PCR以及Western-Blot證實CX43基因修飾后的hUCSCs Cx43表達(dá)水平較未轉(zhuǎn)染組顯著升高(CX43mRNA升高2倍,CX43蛋白表達(dá)升高3倍)(p0.05)。進(jìn)一步檢測GJIC功能,結(jié)果顯示:Ad-Cx43-GFP組細(xì)胞在漂白后40s時的熒光與淬滅前水平基本相當(dāng),而對照組無法恢復(fù)。2.CX43基因修飾的人臍血源基質(zhì)細(xì)胞對L615細(xì)胞的影響:1)以含20%胎牛血清的DMEM+PRMI1640混合培養(yǎng)基作為培養(yǎng)液,成功建立了兩種細(xì)胞的體外接觸共培養(yǎng)體系,共培養(yǎng)72h后,顯微鏡下可見基質(zhì)細(xì)胞與L615細(xì)胞直接接觸,電鏡下能清晰的看到基質(zhì)細(xì)胞粘附、包裹L615細(xì)胞。2)CCK8法檢測細(xì)胞增殖:共培養(yǎng)48h,轉(zhuǎn)染Cx43組L615細(xì)胞增殖受到明顯抑制,直接測得OD值,Cx43轉(zhuǎn)染組明顯小于其余各組,兩兩間比較,Cx43轉(zhuǎn)染組與另外兩組差異均有統(tǒng)計學(xué)意義(p0.05)。提示自共培養(yǎng)48h起,轉(zhuǎn)染Cx43組L615細(xì)胞活力明顯降低,存活率明顯降低(p0.05)。3)流式細(xì)胞儀檢測細(xì)胞凋亡:轉(zhuǎn)染CX43組L615細(xì)胞凋亡率為9.70±0.83%,明顯高于未轉(zhuǎn)染組(7.33±0.74%)和陰性對照組(2.50±0.85%),差異有顯著性(p0.05)。4)流式細(xì)胞儀檢測細(xì)胞周期:Cx43轉(zhuǎn)染組G0/G1期細(xì)胞比例約為80.43%,較陰性對照組(84.43%)降低(P0.05);Cx43轉(zhuǎn)染組S期細(xì)胞比例為10.42%,較陰性對照組(6.38%)升高(P0.05)。G2/M期細(xì)胞比例Cx43轉(zhuǎn)染組(8.52%)與單獨培養(yǎng)組(8.15%)相似(P0.05)。未轉(zhuǎn)染組各細(xì)胞周期的比例與單獨培養(yǎng)組無統(tǒng)計學(xué)差異。5)Western-Blot檢測共培養(yǎng)后L615細(xì)胞caspase3、6、7表達(dá):Cx43轉(zhuǎn)染組caspase3表達(dá)較對照組升高1.8倍(P0.05)。Cx43轉(zhuǎn)染組caspase7表達(dá)較對照組升高7倍(P0.05)。caspase6表達(dá)各組間無明顯差異(P0.05)。3.Ad-Cx43-hUCSCs對自體造血干細(xì)胞移植小鼠殘留白血病的抑制效應(yīng):第0天以L615細(xì)胞經(jīng)尾靜脈注射L615同系小鼠致白血病,細(xì)胞量2×106個/只,于接種+5天即在外周血中查見白血病細(xì)胞,接種+8天骨髓、肝臟、脾臟均查見大量白血病細(xì)胞浸潤,平均存活時間:11±1.71天。第3天經(jīng)腹腔注射環(huán)磷酰胺,200mg/kg,化療后+5天白細(xì)胞明顯降低,化療后+7天白細(xì)胞數(shù)開始回升,+17天接近正常,此時骨髓、肝臟、脾臟均未查見白血病細(xì)胞,于+23天疾病復(fù)發(fā),平均存活時間27.33±2.49天。提示治療后L615模型小鼠能達(dá)到完全緩解,存活時間延長,但體內(nèi)仍有白血病殘留,最后復(fù)發(fā)。1)Ad-Cx43-hUCSCs體內(nèi)分布情況:移植后24小時即在小鼠骨髓、肝臟、脾臟、肺臟均能檢測到Ad-Cx43-hUCSCs,移植+7天,骨髓、肝臟、脾臟中紅色熒光細(xì)胞數(shù)量增加,熒光強(qiáng)度減弱,肺臟中紅色熒光標(biāo)記細(xì)胞減少。移植+14天,骨髓及其他臟器均不能查見紅色熒光標(biāo)記的細(xì)胞。2)移植小鼠血象變化情況:移植后,Ad-Cx43-hUCSCs+BM組小鼠白細(xì)胞從+8天開始、血小板從+11天開始回升速度明顯快于BM組(P0.05)比較差異具有統(tǒng)計學(xué)意義(P0.05)。3)FRAP檢測兩組小鼠骨髓基質(zhì)細(xì)胞的GJIC功能:BM+Ad-Cx43-hUCSCs聯(lián)合移植組,骨髓基質(zhì)細(xì)胞的熒光強(qiáng)度在淬滅后5min內(nèi)恢復(fù)69.33±1.25%,較BM單獨移植組(51.67±1.7%)明顯增高,有統(tǒng)計學(xué)意義(P0.05)。4)人臍血源基質(zhì)細(xì)胞移植骨髓涂片和病理切片的變化:移植+17天,骨髓涂片顯示,Cx43+hUCBDSCs+BM組未查見白血病細(xì)胞浸潤(p0.05),BM組有大量白血病細(xì)胞浸潤(圖11)。骨髓活檢顯示:Cx43+hUCBDSCs+BM組未見白血病細(xì)胞,BM組骨小梁減少,細(xì)胞形態(tài)偏幼稚,成簇、成團(tuán)分布(圖12)。5)各組小鼠移植后28天內(nèi)的生存率:Ad-Cx43-hUCSCs+BM組小鼠平均生存時間為26.9±1.52天,較BM組(23.70±1.99天)延長(p0.05)Ad-Cx43-hUCSCs+BM組小鼠死亡率20%,較BM組35%降低(p0.05)。結(jié)論:1.CX43基因腺病毒可成功轉(zhuǎn)染hUCSCs,Ad-Cx43-hUCSCs的CX43表達(dá)水平上調(diào),GJCI功能增加;2.成功建立人臍血源基質(zhì)細(xì)胞及L615細(xì)胞共培養(yǎng)體系:Ad-Cx43-hUCSCs與L615細(xì)胞株共培養(yǎng)可抑制L615細(xì)胞增殖,上調(diào)L615細(xì)胞caspase3、7表達(dá)水平,促進(jìn)其凋亡;3.Ad-Cx43-hUCSCs聯(lián)合自體造血干細(xì)胞移植后,可歸巢至骨髓、脾臟、肝臟、肺等器官;聯(lián)合移植可促進(jìn)小鼠造血重建;使骨髓基質(zhì)細(xì)胞GJIC功能增強(qiáng),有助于清除殘留白血病,延長移植小鼠生存。
[Abstract]:Background: hematopoietic stem cell transplantation is an effective method for the treatment of acute leukemia. Autologous hematopoietic stem cell transplantation has the advantages of wide application, less cost and high safety, but the recurrence rate of patients after transplantation is high. The root of Minimal Residual Disease, MRD is not thorough. Long, supportive and asylum are important factors for leukemic residues. Stromal cells are the main components of the hematopoietic microenvironment, which regulate the growth and infiltration of leukemic cells, mediate residual and drug resistance of leukemia. Intercellular communication (gap junction intercellular communication, GJIC) is a common direct link between adjacent cells. Connexin43 (connexin43, CX43) mediated GJIC widely exists in bone marrow stromal cells, stromal cells and hematopoietic cells, which is essential for the involvement of bone marrow stroma in hematopoiesis regulation..GJIC weakening or deletion plays an important role in the occurrence and development of solid tumors. The detection of cells showed that the expression of CX43 decreased obviously, the function of GJIC decreased obviously and the inhibition of leukemia weakened. The clinical study found that the GJIC function of the patients after autologous hematopoietic stem cell transplantation was at a low level for a long time, causing the inhibition of the bone marrow microenvironment to leukaemia, which may be easy to relapse in the residual leukemia. Factors. Human umbilical cord blood source stromal cells (human umbilical cord blood-derived stromal cells, hUCSCs) can inhibit the proliferation and promote apoptosis of leukemic cell lines in vitro. In vivo, it can return to the bone marrow to repair the hematopoietic microenvironment. The Cx43 gene transfected into a new type of human umbilical cord stromal cells and autologous hematopoietic stem cells was transplanted to the pretreated small residual disease mouse model. The function of GJIC function after reconstructing and transplantation to purify the small residual disease in mice and reduce the recurrence of white blood disease after autologous transplantation is a scientific idea. The research content of the study on the purification effect and mechanism of residual leukemia after autologous hematopoietic stem cell transplantation (autologous hematopoietic stem cell transplantation, AHSCT) by the National Natural Science Fund Project (No.81070388) "CX43 modifier transplantation of umbilical cord blood stromal cells" (autologous hematopoietic stem cell transplantation, AHSCT). To influence the proliferation and differentiation of leukemic cells to achieve the goal of degradation, to explore new methods of leukemia treatment and to accumulate theoretical and experimental basis. Objective: To observe the transplantation of Cx43 modified human umbilical cord blood stromal cells transplantation in the microenvironment of small residual leukemia mice after hematopoietic stem cell transplantation. And the repair of abnormal bone marrow microenvironment, by repairing and enhancing the GJIC function of bone marrow stroma in mice after transplantation, improving the hematopoietic regulation function of bone marrow microenvironment, and realizing the purification of residual leukemia in mice. Method: 1.CX43 recombinant adenovirus vector (Ad-Cx43-GFP) transfected human umbilical cord stromal cells according to the method established in this department. Human umbilical cord blood was separated and cultured to obtain human umbilical cord stromal cells. Ad-Cx43-GFP recombinant adenovirus vector was constructed and transfected into human umbilical cord stromal cells according to routine methods of our family. Immunofluorescence, PCR, Western-Blot, and light decolorization (fluorescence recovery after photo bleaching, FRAP) were used respectively. After transfection, Cx43 expressed.2. to construct a co culture system of human umbilical cord blood stromal cells and L615 cells. In vitro observation on the regulation effect of human umbilical cord blood derived stromal cells on L615 leukemia cells before and after CX43 transfection: 1) negative control group: L615 cells were cultured separately; 2) hUCSCs/ L615 group: human umbilical cord blood stromal cells and L615 cell co culture group 3) Ad-Cx43-hUCSCs/L615 group: Ad-Cx43-GFP transfected human umbilical cord blood stromal cells and L615 cells co culture group CCK8 method to detect the growth curve of L615 cells after co culture and compare the proliferation rate of L615 cells in each group; flow cytometry was used to detect the apoptosis rate and cell cycle of L615 cells after co culture; Western-Blot detected L615 cell Caspase3 after co culture. The expression of 6,7.3. was used to construct a L615 leukemia MRD mouse model, and the human umbilical cord blood stromal cells were combined with autologous hematopoietic stem cell transplantation. The inhibitory effect of the human umbilical cord blood stromal cells on the residual leukemia in the autologous hematopoietic stem cell transplantation mice after the transfection was injected into the L615 mice by the tail vein of L615 cells, and the intraperitoneal injection of ring phosphorus 3 days later. The L615 leukemia MRD mouse model was obtained. The experiment was divided into two groups: two groups: 1) group BM: bone marrow hematopoietic stem cell transplantation group 2) group Ad-Cx43-hUCSCs+BM: Ad-Cx43-GFP transfected human umbilical cord blood stromal cells combined with bone marrow hematopoietic stem cell transplantation group with residual leukemia L615 as receptor rat, normal L615 as donor and recipient 6 in zeroth days. Gy 60Co gamma ray TBI, 6 h after irradiation, the bone marrow hematopoietic stem cell cells from rat donor and CM-Dil labeled Cx43 modified human umbilical cord blood stromal cells were injected into the tail vein, among which, the bone marrow cells transfused in group BM were 2 x 106, and 1 x 106 / only of each group of bone marrow cells and human umbilical cord stromal cells were transfused in Ad-Cx43-hUCSCs+BM group. The hematopoietic reconstitution was observed after the blood routine examination of the tail vein blood after transplantation. The bone marrow was smeared and the bone marrow pathological sections were examined in different groups of mice after transplantation. The distribution of Ad-Cx43-hUCSCs after transplantation was traced by fluorescent labeling, and the GJIC function of bone marrow stromal cells after transplantation was detected at different time points after transplantation. Pathological section and HE staining of liver, spleen, lung and kidney were taken respectively. The survival rate of mice after transplantation was observed. Results: 1. the recombinant adenovirus vector of Ad-Cx43-GFP was successfully constructed and transfected into human umbilical cord stromal cells: the expression of green fluorescence was seen in 24h after transfection, the 48h fluorescence was reached to the peak, the transfection efficiency was (89.30 + 3.12)%. Immunofluorescence, RT-P CR and Western-Blot confirmed that the expression level of hUCSCs Cx43 after CX43 gene modification was significantly higher than that of untransfected group (CX43mRNA increased 2 times, CX43 protein expression increased by 3 times) (P0.05). The function of GJIC was further detected. The results showed that the fluorescence of Ad-Cx43-GFP group cells in 40s after bleaching was basically equal to that before quenching, while the control group was unable to restore.2.CX. The effect of 43 gene modified human umbilical cord blood stromal cells on L615 cells: 1) the DMEM+PRMI1640 mixed medium containing 20% fetal bovine serum was used as the culture medium, and the co culture system of two kinds of cells was successfully established. After the co culture of 72h, the matrix cells were directly exposed to the L615 cells under the microscope. Under the electron microscope, the matrix could be clearly seen. Cell adhesion, encapsulated L615 cell.2) CCK8 method to detect cell proliferation: co culture 48h, Cx43 group L615 cell proliferation was significantly inhibited, direct measurement of OD value, Cx43 transfection group was significantly smaller than the other groups, 22 comparison, Cx43 transfection group and the other two groups have statistical significance (P0.05). Suggest self culture 48h, Cx43 group L615 finer Cell viability was significantly reduced and survival rate decreased significantly (P0.05).3) flow cytometry was used to detect cell apoptosis: the apoptosis rate of L615 cells in CX43 transfected group was 9.70 + 0.83%, significantly higher than that in untransfected group (7.33 + 0.74%) and negative control group (2.50 + 0.85%), and the difference was significant (P0.05).4) flow cytometry to detect cell cycle: the proportion of G0/G1 phase cells in Cx43 transfected group About 80.43%, compared with the negative control group (84.43%) (P0.05), the proportion of S phase cells in the Cx43 transfection group was 10.42%, compared with the negative control group (6.38%), the ratio of Cx43 transfected group (8.52%) to the single culture group (8.15%) was similar (P0.05). The proportion of cell cycle in the untransfected group was not statistically different from that of the single culture group.5) Western-Blot test. The expression of caspase3,6,7 in L615 cells after co culture: the expression of Caspase3 in Cx43 transfected group was 1.8 times higher than that of the control group (P0.05).Cx43 transfection group, the expression of caspase7 in the transfected group was 7 times higher than that of the control group. There was no significant difference between each group (P0.05).Caspase6 expression (P0.05).3.Ad-Cx43-hUCSCs on the residual leukemia in the autologous hematopoietic stem cell transplantation mice: zeroth days The L615 cells were injected with L615 mice induced by the tail vein to induce leukemia. The cell volume was 2 x 106 / only. The leukemia cells were found in the peripheral blood for +5 days. The infiltration of a large number of leukemic cells was observed on +8 days' bone marrow, liver and spleen. The average survival time was 11 + 1.71 days. Third days by intraperitoneal injection of cyclophosphamide, 200mg/kg, +5 days after chemotherapy. In +7 days after chemotherapy, the number of leukocytes began to recover and the +17 days were close to normal. At this time, the leukemia cells were not found in the bone marrow, liver and spleen, and the disease recurred on +23 days and the average survival time was 27.33 + 2.49 days. It suggested that after the treatment, the L615 model mice were able to complete remission and prolong the survival time, but there were still leukemic residues in the body and finally relapsed. 1) distribution of Ad-Cx43-hUCSCs in vivo: 24 hours after transplantation, Ad-Cx43-hUCSCs was detected in mice bone marrow, liver, spleen and lung, the number of red fluorescent cells in bone marrow, liver and spleen increased, fluorescence intensity decreased, red fluorescent labeled cells in the lungs decreased, and the bone marrow and other organs could not be found red after transplantation for +14 days. Changes of hematogram in mice transplanted with color fluorescent labeled cell.2): after transplantation, the white blood cells in group Ad-Cx43-hUCSCs+BM began to start from +8 days, and the rate of platelet from +11 days was faster than that of BM group (P0.05). The difference was statistically significant (P0.05).3) FRAP detection of GJIC function of bone marrow stromal cells in two groups of mice: BM+Ad-Cx43-hUCSCs Union The fluorescence intensity of bone marrow stromal cells in the transplantation group was 69.33 + 1.25% after quenching and after 5min, which was significantly higher than that in the BM alone group (51.67 + 1.7%), with a statistically significant (P0.05).4) changes in bone marrow smears and pathological sections of human umbilical cord blood stromal cells transplantation: transplantation +17 days, bone marrow smears showed that leukemic cells were not found in the Cx43+hUCBDSCs+BM group. Infiltration (P0.05), group BM with a large number of leukemia cell infiltration (Figure 11). Bone marrow biopsy showed that no leukemia cells in group Cx43+hUCBDSCs+BM, BM group bone trabecula decreased, cell morphology was naive, cluster, group distribution (Figure 12).5) the survival rate of mice in each group after 28 days after transplantation: the average survival time of group Ad-Cx43-hUCSCs+BM mice was 26.9 + 1.52 days, more than BM Group (23.70 + 1.99 days) prolonged (P0.05) Ad-Cx43-hUCSCs+BM group death rate of 20%, compared with group BM 35% (P0.05). Conclusion: 1.CX43 gene adenovirus can successfully transfect hUCSCs, Ad-Cx43-hUCSCs CX43 expression level up, GJCI function increase; 2. successfully establish human umbilical cord blood derived basic cell and L615 cell co culture system: Ad-Cx43-hUCSCs and L615 cells Co culture can inhibit the proliferation of L615 cells, increase the caspase3,7 expression level of L615 cells and promote its apoptosis. 3.Ad-Cx43-hUCSCs combined with autologous hematopoietic stem cell transplantation can return to the bone marrow, spleen, liver, lung and other organs. Combined transplantation can promote the reconstruction of hematopoiesis in mice, enhance the GJIC function of bone marrow stromal cells, and help to clear the residual white blood. Disease, prolonging the survival of transplanted mice.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R733.7

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