CX43修饰hUCSCs输注对自体造血干细胞移植后残留白血病的净化效应及机制

发布时间：2018-06-29 13:49

本文选题：人脐血源基质细胞 + 间隙连接蛋白43　；参考：《第三军医大学》2017年博士论文

【摘要】：背景:造血干细胞移植是治疗急性白血病的有效手段,自体造血干细胞移植具有适用范围广、费用较少、安全性高等优点,但是移植后患者的复发率较高。微小残留病(Minimal Residual Disease,MRD)清除不彻底是其根源。造血微环境对白血病的生长、支持、庇护是白血病残留的重要因素。基质细胞是造血微环境的主要成分,具有调控白血病细胞生长、浸润,介导白血病的残留及耐药的作用。间隙连接细胞间通讯(gap junction intercellular communication,GJIC)是相邻细胞间普遍存在的直接通讯方式。间隙连接蛋白43(connexin43,CX43)介导的GJIC广泛存在于骨髓基质细胞、基质细胞与造血细胞之间,是骨髓基质参与造血调控的必要条件。GJIC减弱或缺失在实体肿瘤的发生发展中起着重要作用。我们前期对急性白血病患者的骨髓基质细胞进行检测发现,其CX43表达明显降低,GJIC功能也明显减弱,对白血病的阻抑作用减弱;临床研究发现,自体造血干细胞移植后患者的GJIC功能长时间处于较低水平,导致骨髓微环境对白血病的阻抑作用减弱,可能是残留白血病易复发的因素。人脐血源基质细胞(human umbilical cord blood-derived stromal cells,hUCSCs)在体外对白血病细胞株具有抑制增殖及促进凋亡的作用,体内能归巢至骨髓修复造血微环境。能否利用人脐血源基质细胞这一基因载体和对白血病细胞具有阻抑作用的特性,将Cx43基因转染新型人脐血源基质细胞与自体造血干细胞联合移植给预处理后的微小残留病小鼠模型,起到重建移植后GJIC功能以净化小鼠微小残留病的作用,从而降低自体移植后白血病的复发是值得验证的科学设想。本课题为国家自然科学基金项目(No.81070388)“CX43修饰人脐血源基质细胞移植对自体造血干细胞移植(autologous hematopoietic stem cell transplantation,AHSCT)后残留白血病净化效应及机制研究”的研究内容。试图从改造白血病造血微环境功能GJIC角度去影响白血病细胞的增殖、分化过程,从而达到去恶化的目的,为探寻白血病治疗的新方法,积累理论和实验依据奠定基础。目的:观察Cx43修饰的新型人脐血源基质细胞移植在造血干细胞移植后的微小残留白血病小鼠骨髓微环境的定植和对异常骨髓微环境的修复作用,通过修复和增强移植后小鼠骨髓基质GJIC功能,改善骨髓微环境造血调控功能,实现对小鼠残留白血病体内净化。方法:1.CX43重组腺病毒载体(Ad-Cx43-GFP)转染人脐血源基质细胞按本科室已经建立的方法对健康产妇脐带血进行细胞分离并贴壁培养,获得人脐血源基质细胞。按我科常规方法构建Ad-Cx43-GFP重组腺病毒载体并转染人脐血源基质细胞。分别用免疫荧光法、PCR、Western-Blot、光脱色荧光恢复技术(fluorescence recovery after photo bleaching,FRAP)检测转染后Cx43表达。2.构建人脐血源基质细胞及L615细胞共培养体系,体外观察CX43转染前后人脐血源基质细胞对L615白血病细胞的调节作用实验分三组:1)阴性对照组:L615细胞单独培养;2)hUCSCs/L615组:人脐血源基质细胞与L615细胞共培养组3)Ad-Cx43-hUCSCs/L615组:Ad-Cx43-GFP转染的人脐血源基质细胞与L615细胞共培养组CCK8法检测共培养后L615细胞的生长曲线,对比各组间L615细胞增殖率;流式细胞术检测共培养后L615细胞的凋亡率及细胞周期;Western-Blot检测共培养后L615细胞caspase3、6、7表达情况。3.构建L615白血病MRD小鼠模型,进行人脐血源基质细胞联合自体造血干细胞移植,观察转染后人脐血源基质细胞对自体造血干细胞移植小鼠残留白血病的抑制效应将L615细胞经尾静脉注入L615同系小鼠,3天后经腹腔注射环磷酰胺200mg/kg,从而获得L615白血病MRD小鼠模型。实验分为两组:1)BM组:骨髓造血干细胞移植组2)Ad-Cx43-hUCSCs+BM组:Ad-Cx43-GFP转染的人脐血源基质细胞联合骨髓造血干细胞移植组以残留白血病L615为受体鼠,以正常L615为供鼠,受鼠在第0天接受6.0 Gy 60Coγ射线TBI;照射后6 h,经尾静脉注射来自供鼠的骨髓造血干细胞细胞以及CM-Dil标记的Cx43修饰的人脐血源基质细胞,其中,BM组输注骨髓细胞为2×106个/只、Ad-Cx43-hUCSCs+BM组输注骨髓细胞及人脐血源基质细胞各1×106个/只。对不同组小鼠移植后尾静脉血行血常规检查以观察造血重建情况;对不同组小鼠移植后的骨髓进行涂片及骨髓病理切片检查;利用荧光标记追踪Ad-Cx43-hUCSCs移植后体内分布情况;对移植后小鼠的骨髓基质细胞进行GJIC功能检测,于移植后不同时间点分别取肝脏、脾脏、肺、肾脏进行病理切片及HE染色;观察各组小鼠移植后的生存率。结果:1.成功构建Ad-Cx43-GFP重组腺病毒载体并转染人脐血源基质细胞:转染后24h可见绿色荧光表达,48h荧光表达达到峰值,转染效率(89.30±3.12)%。免疫荧光、RT-PCR以及Western-Blot证实CX43基因修饰后的hUCSCs Cx43表达水平较未转染组显著升高(CX43mRNA升高2倍,CX43蛋白表达升高3倍)(p0.05)。进一步检测GJIC功能,结果显示:Ad-Cx43-GFP组细胞在漂白后40s时的荧光与淬灭前水平基本相当,而对照组无法恢复。2.CX43基因修饰的人脐血源基质细胞对L615细胞的影响:1)以含20%胎牛血清的DMEM+PRMI1640混合培养基作为培养液,成功建立了两种细胞的体外接触共培养体系,共培养72h后,显微镜下可见基质细胞与L615细胞直接接触,电镜下能清晰的看到基质细胞粘附、包裹L615细胞。2)CCK8法检测细胞增殖:共培养48h,转染Cx43组L615细胞增殖受到明显抑制,直接测得OD值,Cx43转染组明显小于其余各组,两两间比较,Cx43转染组与另外两组差异均有统计学意义(p0.05)。提示自共培养48h起,转染Cx43组L615细胞活力明显降低,存活率明显降低(p0.05)。3)流式细胞仪检测细胞凋亡:转染CX43组L615细胞凋亡率为9.70±0.83%,明显高于未转染组(7.33±0.74%)和阴性对照组(2.50±0.85%),差异有显著性(p0.05)。4)流式细胞仪检测细胞周期:Cx43转染组G0/G1期细胞比例约为80.43%,较阴性对照组(84.43%)降低(P0.05);Cx43转染组S期细胞比例为10.42%,较阴性对照组(6.38%)升高(P0.05)。G2/M期细胞比例Cx43转染组(8.52%)与单独培养组(8.15%)相似(P0.05)。未转染组各细胞周期的比例与单独培养组无统计学差异。5)Western-Blot检测共培养后L615细胞caspase3、6、7表达:Cx43转染组caspase3表达较对照组升高1.8倍(P0.05)。Cx43转染组caspase7表达较对照组升高7倍(P0.05)。caspase6表达各组间无明显差异(P0.05)。3.Ad-Cx43-hUCSCs对自体造血干细胞移植小鼠残留白血病的抑制效应:第0天以L615细胞经尾静脉注射L615同系小鼠致白血病,细胞量2×106个/只,于接种+5天即在外周血中查见白血病细胞,接种+8天骨髓、肝脏、脾脏均查见大量白血病细胞浸润,平均存活时间:11±1.71天。第3天经腹腔注射环磷酰胺,200mg/kg,化疗后+5天白细胞明显降低,化疗后+7天白细胞数开始回升,+17天接近正常,此时骨髓、肝脏、脾脏均未查见白血病细胞,于+23天疾病复发,平均存活时间27.33±2.49天。提示治疗后L615模型小鼠能达到完全缓解,存活时间延长,但体内仍有白血病残留,最后复发。1)Ad-Cx43-hUCSCs体内分布情况:移植后24小时即在小鼠骨髓、肝脏、脾脏、肺脏均能检测到Ad-Cx43-hUCSCs,移植+7天,骨髓、肝脏、脾脏中红色荧光细胞数量增加,荧光强度减弱,肺脏中红色荧光标记细胞减少。移植+14天,骨髓及其他脏器均不能查见红色荧光标记的细胞。2)移植小鼠血象变化情况:移植后,Ad-Cx43-hUCSCs+BM组小鼠白细胞从+8天开始、血小板从+11天开始回升速度明显快于BM组(P0.05)比较差异具有统计学意义(P0.05)。3)FRAP检测两组小鼠骨髓基质细胞的GJIC功能:BM+Ad-Cx43-hUCSCs联合移植组,骨髓基质细胞的荧光强度在淬灭后5min内恢复69.33±1.25%,较BM单独移植组(51.67±1.7%)明显增高,有统计学意义(P0.05)。4)人脐血源基质细胞移植骨髓涂片和病理切片的变化:移植+17天,骨髓涂片显示,Cx43+hUCBDSCs+BM组未查见白血病细胞浸润(p0.05),BM组有大量白血病细胞浸润(图11)。骨髓活检显示:Cx43+hUCBDSCs+BM组未见白血病细胞,BM组骨小梁减少,细胞形态偏幼稚,成簇、成团分布(图12)。5)各组小鼠移植后28天内的生存率:Ad-Cx43-hUCSCs+BM组小鼠平均生存时间为26.9±1.52天,较BM组(23.70±1.99天)延长(p0.05)Ad-Cx43-hUCSCs+BM组小鼠死亡率20%,较BM组35%降低(p0.05)。结论:1.CX43基因腺病毒可成功转染hUCSCs,Ad-Cx43-hUCSCs的CX43表达水平上调,GJCI功能增加;2.成功建立人脐血源基质细胞及L615细胞共培养体系:Ad-Cx43-hUCSCs与L615细胞株共培养可抑制L615细胞增殖,上调L615细胞caspase3、7表达水平,促进其凋亡;3.Ad-Cx43-hUCSCs联合自体造血干细胞移植后,可归巢至骨髓、脾脏、肝脏、肺等器官;联合移植可促进小鼠造血重建;使骨髓基质细胞GJIC功能增强,有助于清除残留白血病,延长移植小鼠生存。
[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.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R733.7

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