人脐血源基质细胞新型微环境对残留白血病细胞的作用及机制探讨
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摘要
残留白血病细胞或称白血病微小残留病(minimal residual disease;MRD)是白血病治疗达到完全缓解后体内残留的白血病细胞,多处于G0期,对化疗药物不敏感,是白血病难治、复发的主要根源。目前,尚未发现真正的白血病细胞特异性抗原,虽然采用多种方法联合但均不能满意杀灭和有效清除患者体内的MRD,而且采用化疗新药和更强烈的联合化疗方案多对正常造血功能造成严重的损伤。针对MRD,目前尚缺乏有效低毒的清除措施,已成为严重束缚白血病治疗取得突破性进展的关键性障碍之一。探索不同来源基质细胞所构建造血微环境对MRD的作用及机制,对进一步降低白血病复发、提高白血病长期生存率,具有重要的理论和实践意义。
造血微环境(hematopoietic inductive microenvironment, HIM)是调控造血干/祖细胞生长发育的“土壤”,骨髓基质细胞(bone marrow stromal cells, BMSCs)是构成造血微环境的重要成分。白血病细胞是恶性克隆性增殖的造血细胞,其生存、分化、增殖同样依赖HIM的调控,白血病骨髓基质细胞微环境有助于白血病细胞的庇护、生长和逃避化疗药物的杀伤,是MRD产生的重要机制之一,因此从造血微环境入手探索清除MRD的有效方法是白血病治疗的新的切入点。
本课题组的既往研究证实:在适当的细胞因子组合的条件下,可成功培养、扩增人脐血来源的基质细胞(human umbilical cord blood-derived stromal cells, hUCBDSCs);扩增后的hUCBDSCs在细胞组成和免疫表型上与人骨髓基质细胞(human bone marrow stromal cells, hBMSCs)相似,可以分泌多种细胞因子,具有与hBMSCs相似的支持和重建造血功能的物质基础,可称为人脐血源基质细胞造血微环境(hematopoietic inductive microenvironment from human umbilical cord blood-derived stromal cells, hUCBDSC-HIM)。
人急性T淋巴细胞白血病细胞株Jurkat细胞分别与hUCBDSC-HIM和正常人骨髓基质细胞造血微环境(hBMSC-HIM)共培养发现:hUCBDSC-HIM具有较hBMSC-HIM对Jurkat细胞更强的抑制作用和促凋亡作用。
鉴于此,本实验将系统、深入地研究不同来源基质细胞所模拟造血微环境对残留耐药Jurkat细胞的细胞周期分布、增殖、分化、凋亡及药物敏感性等生物学特性的影响,并对其机制进行初步探讨。
研究内容及方法:
1.不同来源骨髓基质细胞微环境对Jurkat细胞生物学特性的影响
实验分组:①Jurkat细胞悬浮培养组;②Jurkat细胞/正常hBMSCs共培养组;③Jurkat细胞/白血病hBMSCs共培养组。
倒置显微镜和扫描电镜观察Jurkat细胞与BMSCs的位相关系;MTT法检测Jurkat细胞的黏附率;Transwell法检测Jurkat细胞迁移侵袭功能变化;CCK-8法、Real-time Q-PCR法、Western blot法检测Jurkat细胞增殖特性的变化;流式细胞仪法检测Jurkat细胞周期及DNR摄药量变化;流式细胞仪法、透射电镜、Real-time Q-PCR法、Western blot法检测Jurkat细胞的凋亡;NBT还原法检测Jurkat细胞分化水平变化。
2.残留耐药Jurkat细胞的培养及其耐药机制的初步探讨
体外分离培养初发急性淋巴细胞白血病患者BMSCs以模拟骨髓造血微环境,在与Jurkat细胞长期共培养的过程中选用含DNR 50ng/ml的培养液进行培养,随着培养时间的延长,绝大部分Jurkat细胞漂浮死亡。极少数残留的Jurkat细胞逐渐恢复活力并获得抗药能力,可以在DNR终浓度为50ng/ml的培养液中存活、增殖,将该细胞记为Jurkat/DNR细胞。
CCK-8法检测Jurkat及Jurkat/DNR细胞的增殖特性及对多种化疗药物的耐药系数;流式细胞仪法检测Jurkat及Jurkat/DNR细胞周期及DNR摄药量;通过检测Caspase-Glo 3/7活性、Real-time Q-PCR法、Western blot法检测Jurkat及Jurkat/DNR细胞的凋亡相关指标;Real-time Q-PCR法检测Jurkat及Jurkat/DNR细胞多药耐药相关基因:MDR1及MRP mRNA的表达。
3.人脐血源基质细胞对残留耐药Jurkat细胞的作用
实验分组:①Jurkat/DNR细胞悬浮培养组;②Jurkat/DNR细胞/正常hBMSCs共培养组;③Jurkat/DNR细胞/hUCBDSCs共培养组;④Jurkat/DNR细胞/白血病hBMSCs共培养组(仅在检测对IDA敏感性时设定)。
收集悬浮培养及共培养10d的Jurkat/DNR细胞,检测其增殖、细胞周期分布、凋亡、分化、对IDA药物敏感性及多药耐药基因MRD1 mRNA表达水平的变化。
结果:
1.倒置显微镜及扫描电镜观察:随培养时间延长,正常hBMSCs及白血病hBMSCs黏附、龛合、包裹Jurkat细胞,对共培养的Jurkat细胞具有一定的屏蔽作用,在一定程度上影响其生物学特性:
1.1黏附、迁移作用:白血病hBMSCs对Jurkat细胞的黏附、趋化作用强于正常hBMSCs;
1.2增殖曲线:正常hBMSCs对Jurkat细胞的增殖抑制作用强于白血病hBMSCs;
1.3细胞周期分布:白血病hBMSCs共培养组Jurkat细胞G0/G1+G2/M期细胞比例明显增高;正常hBMSCs共培养组S+G2/M期细胞比例明显增高;
1.4凋亡:白血病hBMSCs抑制Jurkat细胞凋亡,而正常hBMSCs促Jurkat细胞凋亡;
1.5细胞分化:白血病hBMSCs对共培养Jurkat细胞的促分化作用弱于正常hBMSCs;
1.6对DNR的摄药量:共培养组Jurkat细胞的摄药量显著低于悬浮培养组,其中白血病hBMSCs共培养组Jurkat细胞的摄药量最低。
2嵌合于基质细胞层中的Jurkat细胞在DNR的持续刺激作用下逐步产生耐药性,获得残留耐DNR的Jurkat细胞。
Jurkat/DNR细胞与Jurkat细胞相比有以下特点:
2.1增殖曲线:Jurkat/DNR细胞较Jurkat细胞的增殖速度明显减缓
2.2细胞周期分布:G0/G1期细胞比例增高;异二倍体细胞比例增高;
2.3凋亡:发生自发凋亡的细胞明显减少;
2.4 DNR摄药量:单个Jurkat/DNR细胞内平均药物浓度明显减低;
2.5对多种化疗药物产生多药耐药;
2.6多药耐药相关基因:Jurkat/DNR细胞内可检测到MDR1 mRNA表达;MRP mRNA表达水平升高。
3.人脐血源基质细胞对残留耐药Jurkat/DNR细胞的作用
3.1增殖曲线:hUCBDSCs对Jurkat/DNR细胞的增殖抑制作用强于正常hBMSCs;
3.2细胞周期分布:hUCBDSCs及正常hBMSCs均可促Jurkat/DNR细胞进入细胞周期,hUCBDSCs共培养组G0/G1期细胞比例最低;共培养组Jurkat/DNR细胞中异二倍体比例显著降低;
3.3凋亡和分化:hUCBDSCs对共培养Jurkat/DNR细胞自发凋亡及分化的促进作用均强于正常hBMSCs;
3.4对IDA的敏感性:hUCBDSCs及hBMSCs均可提高Jurkat/DNR对IDA的敏感性,而hUCBDSCs要优于hBMSCs;
3.5 MDR1 mRNA的表达:共培养组Jurkat/DNR细胞MDR1 mRNA表达水平低于悬浮培养组,其中hUCBDSCs共培养组的表达水平最低。
结论:
1.白血病骨髓基质细胞微环境有助于白血病细胞的庇护、生长和逃避化疗药物的杀伤;正常骨髓源基质细胞较白血病源骨髓基质细胞对Jurkat细胞具有更强的促凋亡、分化及抑制增殖的作用。同时,正常hBMSCs可以增加Jurkat细胞对DNR的敏感性。
2.白血病骨髓基质细胞模拟的造血微环境可诱导Jurakt细胞产生多药耐药,是残留耐药形成的一个重要原因,提示可能通过改造白血病患者造血微环境以抑制残留白血病的形成及减少复发。
3.骨髓源基质细胞和脐血源基质细胞均可改造白血病造血微环境,在一定程度上逆转Jurkat/DNR细胞的耐药性。
4.脐血源基质细胞对白血病造血微环境的改造作用及逆转Jurkat/DNR细胞耐药的作用更强。
Minimal residual disease (MRD) is the residual leukemia cells in vivo after complete remission of leukemia, and the cells are mostly in the G0 phase and not sensitive to chemotherapeutic drugs, which is the major source of refractoriness and relapse for leukemia. At present, we have not yet found a exact leukemic cell-specific antigen. Although we have applied a number of ways collaborated but the kill and remove effect of the MRD can not be satisfied and effective, and the application of chemotherapy drugs and a stronger collaborated chemotherapy brings serious damage to the normal hematopoietic function. For the removal of MRD, we are still lack of an effective and low toxicity method, which has become one of the serious key obstacles for the breakthrough of the treatment of leukemia. To explore the effect and mechanism of hemopoietic microenvironment built by matrix cells from different sources to the MRD has an important theoretical and practical significance for further reducing leukemia relapse and improving long-term survival rate of leukemia.
Hematopoietic inductive microenvironment(HIM),which was mainly composed of bone marrow stromal cells(BMSCs), acted as the soil in regulating the growth and development of hematopoietic stem/progenitor cell.The development and proliferation of leukemic cells were dependent on the abnormal HIM.The BMSCs derived from leukemia that supported and protected leukemic cells involved in the occurrence and refractory of leukemia. One of the important mechanisms for the supply of MRD is that BMSCs can help leukemia cells to evade the chemotherapy drugs, so that to explore an effective way to clear MRD starting from the hematopoietic microenvironment is a new entry point.
The group's past research has shown that: under the condition of the appropriate combination of cytokines, human umbilical cord blood-derived stromal cells (hUCBDSCs ) can be successfully cultivated and amplified; after the amplification, the cells composition and immunophenotype of the hUCBDSCs is similar to that of hBMSCs, which can secrete a variety of cytokines and carries the similar material basis for supporting and reconstructing the hematopoietic function with hBMSCs, so that it may be called hematopoietic inductive microenvironment from human umbilical cord blood-derived stromal cells (hUCBDSC-HIM).
The co-culture of human acute T Lymphocytic leukemia cell line Jurkat cells respectively with hUCBDSC-HIM and hBMSC-HIM has found: hUCBDSC-HIM has a stronger inhibition and the phenomenon of apoptosis promotion to the Jurkat cells compared with hBMSC-HIM.
Based on this point, the experiment will study the influences of hemopoietic microenvironment simulated by stromal cells from different sources to the biological characteristics of residual drug resistant Jurkat cells, such as generation cycle disposition, proliferation, differentiation, apoptosis and drug sensitivity systematically and in-depth, and carry on the preliminary study of its mechanisms.
Contents and methods:
1. Influence of microenvironment of normal and acute lymphoblastic leukemia bone marrow stromal cells to Jurkat cells’biological characteristics respectively
Groups: Jurkat suspended culture group; Jurkat/nomal hBMSCs co-culture group; Jurkat leukemic hBMSCs co-culture group.
The spatial relationship between the Jurkat cells and stromal cells was observed under the inverted microscope and scanning electron microscope. MTT method for the detection of the adhesion rate of Jurkat cell; Transwell method for the detection of Jurkat cell migration and invasion function changes; CCK-8 method, Real-time Q-PCR method, Western blot method for the detection of Jurkat cell proliferation characteristics change; flow cytometry for the detection of Jurkat cell cycle and DNR dose perturbation changes; flow cytometry method, transmission electron microscopy, Real-time Q-PCR method, Western blot method for the detection of Jurkat cells apoptosis; NBT reduction method for the detection of Jurkat cell differentiation level changes.
2. Preliminary study on the culture and its drug resistance mechanism of residual drug resistance Jurkat cell
We use the isolated culture of BMSCs in patients with primary acute lymphoblastic leukemia in vitro to simulate the bone marrow hematopoietic microenvironment. In the long process of co-culture with Jurkat cells, we choose the culture medium containing 50ng/ml DNR. With the culture time extended, the vast majority of Jurkat cells are floating dead. A very small number of residual Jurkat cells recover and got the drug resistant ability that can survive and proliferate in the culture medium with 50ng/ml final concentration of DNR, these cells are recorded as Jurkat / DNR cells. We use CCK-8 method to detect the proliferation characteristics and the resistance coefficient of a wide range of chemotherapy drug of Jurkat and Jurkat / DNR cell; flow cytometry method for the detection of generation cycle and the dose perturbation of the DNR for Jurkat and Jurkat / DNR cell; through the detection of Caspase - Glo 3 / 7 activity, Real-time Q-PCR method, Western blot method for the detection of apoptosis-related indicators of Jurkat and Jurkat / DNR cell; Real-time Q-PCR method for multidrug resistance-associated gene of Jurkat and Jurkat / DNR cell : expression of MDR1 and MRP mRNA.
3. The influences of hUCBDSCs to the residual drug resistant Jurkat cells
Groups: Jurkat / DNR cell suspension culture group; Jurkat / DNR cells / normal hBMSCs co-culture group; Jurkat / DNR cells / hUCBDSCs co-culture group; Jurkat / DNR cells / leukemia hBMSCs co-culture group (set only sensitive to IDA). Collect the Jurkat / DNR cells after suspension culture and co-cultured 10 days to detect their changes in proliferation, generation cycle disposition, apoptosis, differentiation, drug sensitivity of the IDA and the expression level of multidrug resistance gene MRD1 mRNA.
Results:
1. Inverted microscope and scanning electron microscopy: With the culture time extended, the normal hBMSCs and leukemia hBMSCs adhere to, combine with, and wrap the Jurkat cells, and they have the shielding function to the co-cultured Jurkat cells, and they will affect its biological characteristics in a certain extent.
1.1 Adhesion and migration function: the adhesion and chemotaxis function to Jurkat cells of leukemia hBMSCs is stronger than normal hBMSCs.
1.2 Inhibition of proliferation: Inhibition of of Jurkat cell’s proliferation by normal hBMSCs is stronger than leukemia hBMSCs.
1.3 Generation cycle disposition: compared with the Jurkat cells in suspension culture, the Jurkat cells proportion in G0/G1 + G2 / M phase of the hBMSCs leukemia cells co-cultured group has significantly increased; the proportion of cells in S + G2 / M phase in the normal hBMSCs co-culture group has significantly increased.
1.4 For the Jurkat cell apoptosis: hBMSCs leukemia inhibit Jurkat cell apoptosis, while normal hBMSCs promote Jurkat cell apoptosis.
1.5 For the Jurkat cell differentiation: the promote-differentiation function of hBMSCs leukemia to the Jurkat cells co-cultured is weaker than normal hBMSCs.
1.6 For the DNR dose changes: the dose of Jurkat cells in the co-cultured prostate group was significantly lower than the suspension culture group, of which Jurkat cells in the hBMSCs leukemia co-culture group is the lowest.
2. Jurkat cells peg-and-socket joined in matrix cell gradually get the drug resistance continuously stimulated by the DNR, left the DNR resistance of Jurkat cells. Compared with Jurkat cells, Jurkat / DNR cells has the following characteristics:
2.1 Proliferation curve: the proliferation speed of Jurkat / DNR cells significantly slow down than Jurkat cells.
2.2 Generation cycle disposition: the proportion of cells in G0/G1 phase increased; the proportion of different diploid cells increased.
2.3 Apoptosis rate: the occurrence of spontaneous apoptosis of cells significantly reduced.
2.4 DNR dosage: the average drug concentration in a single Jurkat / DNR cell markedly reduced.
2.5 Multi-drug resistance to a wide range of chemotherapy drug.
2.6 Multi-drug resistance-associated gene expression: in the Jurkat / DNR cells, we can detect the expression of MDR1 mRNA; its expression level of MRP mRNA increased.
3. hUCBDSCs to the residual drug resistanct Jurkat cells
3.1 Proliferation curve: inhibition of cell proliferation of hUCBDSCs to Jurkat / DNR cells is stronger than normal hBMSCs.
3.2 Generation cycle disposition: hBMSCs and normal hUCBDSCs can both promote Jurkat / DNR cells to the generation cycle, the proportion of cells in G0/G1 phase of hUCBDSCs co-culture group is the lowest; the proportion of different diploid cells in Jurkat / DNR cells in the co-culture group significantly reduce.
3.3 To the cell apoptosis and differentiation: the promotion of hUCBDSCs to the spontaneous apoptosis and differentiation of co-cultured Jurkat / DNR cell is stronger than normal hBMSCs.
3.4 Sensitivity to IDA: hUCBDSCs and hBMSCs can increase the IDA sensitivity of Jurkat / DNR, and hUCBDSCs is superior to hBMSCs.
3.5 mRNA expression in MDR1: the expression of MDR1 mRNA level in co-culture group of Jurkat / DNR cell is lower than the suspension culture group, of which the expression level of the hUCBDSCs co-culture group is the lowest.
Conclusion:
1. Leukemia bone marrow stromal cells micro-environment can contribute to the shelter, growth and evasion of the anti-drug chemotherapy of leukemic cell; compared with Leukemia bone marrow stromal cells, normal bone marrow stromal cells has a stronger function in pro-apoptotic, differentiation and inhibit proliferation to Jurkat cells . At the same time, it can increase the sensitivity of the DNR of Jurkat cells.
2. The hematopoietic micro-environment simulated by leukemia bone marrow stromal cells can induce Jurakt cells to form multi-drug resistance, which is an important reason for the formation of residual drug resistance, suggesting that it is possible to inhibit the formation of residual leukemia and reduced recurrence by transforming the hematopoietic microenvironment in patients with leukemia.
3. Bone marrow-derived stromal cells and cord blood-derived stromal cells can both transform the hematopoietic microenvironment of leukemia, and reverse the drug resistance of Jurkat / DNR cells in a certain extent.
4. Cord blood-derived stromal cells have a stronger effect in the transformation of hematopoietic microenvironment of leukemia and the reverse of Jurkat / DNR cell drug resistance.
造血微环境(hematopoietic inductive microenvironment, HIM)是调控造血干/祖细胞生长发育的“土壤”,骨髓基质细胞(bone marrow stromal cells, BMSCs)是构成造血微环境的重要成分。白血病细胞是恶性克隆性增殖的造血细胞,其生存、分化、增殖同样依赖HIM的调控,白血病骨髓基质细胞微环境有助于白血病细胞的庇护、生长和逃避化疗药物的杀伤,是MRD产生的重要机制之一,因此从造血微环境入手探索清除MRD的有效方法是白血病治疗的新的切入点。
本课题组的既往研究证实:在适当的细胞因子组合的条件下,可成功培养、扩增人脐血来源的基质细胞(human umbilical cord blood-derived stromal cells, hUCBDSCs);扩增后的hUCBDSCs在细胞组成和免疫表型上与人骨髓基质细胞(human bone marrow stromal cells, hBMSCs)相似,可以分泌多种细胞因子,具有与hBMSCs相似的支持和重建造血功能的物质基础,可称为人脐血源基质细胞造血微环境(hematopoietic inductive microenvironment from human umbilical cord blood-derived stromal cells, hUCBDSC-HIM)。
人急性T淋巴细胞白血病细胞株Jurkat细胞分别与hUCBDSC-HIM和正常人骨髓基质细胞造血微环境(hBMSC-HIM)共培养发现:hUCBDSC-HIM具有较hBMSC-HIM对Jurkat细胞更强的抑制作用和促凋亡作用。
鉴于此,本实验将系统、深入地研究不同来源基质细胞所模拟造血微环境对残留耐药Jurkat细胞的细胞周期分布、增殖、分化、凋亡及药物敏感性等生物学特性的影响,并对其机制进行初步探讨。
研究内容及方法:
1.不同来源骨髓基质细胞微环境对Jurkat细胞生物学特性的影响
实验分组:①Jurkat细胞悬浮培养组;②Jurkat细胞/正常hBMSCs共培养组;③Jurkat细胞/白血病hBMSCs共培养组。
倒置显微镜和扫描电镜观察Jurkat细胞与BMSCs的位相关系;MTT法检测Jurkat细胞的黏附率;Transwell法检测Jurkat细胞迁移侵袭功能变化;CCK-8法、Real-time Q-PCR法、Western blot法检测Jurkat细胞增殖特性的变化;流式细胞仪法检测Jurkat细胞周期及DNR摄药量变化;流式细胞仪法、透射电镜、Real-time Q-PCR法、Western blot法检测Jurkat细胞的凋亡;NBT还原法检测Jurkat细胞分化水平变化。
2.残留耐药Jurkat细胞的培养及其耐药机制的初步探讨
体外分离培养初发急性淋巴细胞白血病患者BMSCs以模拟骨髓造血微环境,在与Jurkat细胞长期共培养的过程中选用含DNR 50ng/ml的培养液进行培养,随着培养时间的延长,绝大部分Jurkat细胞漂浮死亡。极少数残留的Jurkat细胞逐渐恢复活力并获得抗药能力,可以在DNR终浓度为50ng/ml的培养液中存活、增殖,将该细胞记为Jurkat/DNR细胞。
CCK-8法检测Jurkat及Jurkat/DNR细胞的增殖特性及对多种化疗药物的耐药系数;流式细胞仪法检测Jurkat及Jurkat/DNR细胞周期及DNR摄药量;通过检测Caspase-Glo 3/7活性、Real-time Q-PCR法、Western blot法检测Jurkat及Jurkat/DNR细胞的凋亡相关指标;Real-time Q-PCR法检测Jurkat及Jurkat/DNR细胞多药耐药相关基因:MDR1及MRP mRNA的表达。
3.人脐血源基质细胞对残留耐药Jurkat细胞的作用
实验分组:①Jurkat/DNR细胞悬浮培养组;②Jurkat/DNR细胞/正常hBMSCs共培养组;③Jurkat/DNR细胞/hUCBDSCs共培养组;④Jurkat/DNR细胞/白血病hBMSCs共培养组(仅在检测对IDA敏感性时设定)。
收集悬浮培养及共培养10d的Jurkat/DNR细胞,检测其增殖、细胞周期分布、凋亡、分化、对IDA药物敏感性及多药耐药基因MRD1 mRNA表达水平的变化。
结果:
1.倒置显微镜及扫描电镜观察:随培养时间延长,正常hBMSCs及白血病hBMSCs黏附、龛合、包裹Jurkat细胞,对共培养的Jurkat细胞具有一定的屏蔽作用,在一定程度上影响其生物学特性:
1.1黏附、迁移作用:白血病hBMSCs对Jurkat细胞的黏附、趋化作用强于正常hBMSCs;
1.2增殖曲线:正常hBMSCs对Jurkat细胞的增殖抑制作用强于白血病hBMSCs;
1.3细胞周期分布:白血病hBMSCs共培养组Jurkat细胞G0/G1+G2/M期细胞比例明显增高;正常hBMSCs共培养组S+G2/M期细胞比例明显增高;
1.4凋亡:白血病hBMSCs抑制Jurkat细胞凋亡,而正常hBMSCs促Jurkat细胞凋亡;
1.5细胞分化:白血病hBMSCs对共培养Jurkat细胞的促分化作用弱于正常hBMSCs;
1.6对DNR的摄药量:共培养组Jurkat细胞的摄药量显著低于悬浮培养组,其中白血病hBMSCs共培养组Jurkat细胞的摄药量最低。
2嵌合于基质细胞层中的Jurkat细胞在DNR的持续刺激作用下逐步产生耐药性,获得残留耐DNR的Jurkat细胞。
Jurkat/DNR细胞与Jurkat细胞相比有以下特点:
2.1增殖曲线:Jurkat/DNR细胞较Jurkat细胞的增殖速度明显减缓
2.2细胞周期分布:G0/G1期细胞比例增高;异二倍体细胞比例增高;
2.3凋亡:发生自发凋亡的细胞明显减少;
2.4 DNR摄药量:单个Jurkat/DNR细胞内平均药物浓度明显减低;
2.5对多种化疗药物产生多药耐药;
2.6多药耐药相关基因:Jurkat/DNR细胞内可检测到MDR1 mRNA表达;MRP mRNA表达水平升高。
3.人脐血源基质细胞对残留耐药Jurkat/DNR细胞的作用
3.1增殖曲线:hUCBDSCs对Jurkat/DNR细胞的增殖抑制作用强于正常hBMSCs;
3.2细胞周期分布:hUCBDSCs及正常hBMSCs均可促Jurkat/DNR细胞进入细胞周期,hUCBDSCs共培养组G0/G1期细胞比例最低;共培养组Jurkat/DNR细胞中异二倍体比例显著降低;
3.3凋亡和分化:hUCBDSCs对共培养Jurkat/DNR细胞自发凋亡及分化的促进作用均强于正常hBMSCs;
3.4对IDA的敏感性:hUCBDSCs及hBMSCs均可提高Jurkat/DNR对IDA的敏感性,而hUCBDSCs要优于hBMSCs;
3.5 MDR1 mRNA的表达:共培养组Jurkat/DNR细胞MDR1 mRNA表达水平低于悬浮培养组,其中hUCBDSCs共培养组的表达水平最低。
结论:
1.白血病骨髓基质细胞微环境有助于白血病细胞的庇护、生长和逃避化疗药物的杀伤;正常骨髓源基质细胞较白血病源骨髓基质细胞对Jurkat细胞具有更强的促凋亡、分化及抑制增殖的作用。同时,正常hBMSCs可以增加Jurkat细胞对DNR的敏感性。
2.白血病骨髓基质细胞模拟的造血微环境可诱导Jurakt细胞产生多药耐药,是残留耐药形成的一个重要原因,提示可能通过改造白血病患者造血微环境以抑制残留白血病的形成及减少复发。
3.骨髓源基质细胞和脐血源基质细胞均可改造白血病造血微环境,在一定程度上逆转Jurkat/DNR细胞的耐药性。
4.脐血源基质细胞对白血病造血微环境的改造作用及逆转Jurkat/DNR细胞耐药的作用更强。
Minimal residual disease (MRD) is the residual leukemia cells in vivo after complete remission of leukemia, and the cells are mostly in the G0 phase and not sensitive to chemotherapeutic drugs, which is the major source of refractoriness and relapse for leukemia. At present, we have not yet found a exact leukemic cell-specific antigen. Although we have applied a number of ways collaborated but the kill and remove effect of the MRD can not be satisfied and effective, and the application of chemotherapy drugs and a stronger collaborated chemotherapy brings serious damage to the normal hematopoietic function. For the removal of MRD, we are still lack of an effective and low toxicity method, which has become one of the serious key obstacles for the breakthrough of the treatment of leukemia. To explore the effect and mechanism of hemopoietic microenvironment built by matrix cells from different sources to the MRD has an important theoretical and practical significance for further reducing leukemia relapse and improving long-term survival rate of leukemia.
Hematopoietic inductive microenvironment(HIM),which was mainly composed of bone marrow stromal cells(BMSCs), acted as the soil in regulating the growth and development of hematopoietic stem/progenitor cell.The development and proliferation of leukemic cells were dependent on the abnormal HIM.The BMSCs derived from leukemia that supported and protected leukemic cells involved in the occurrence and refractory of leukemia. One of the important mechanisms for the supply of MRD is that BMSCs can help leukemia cells to evade the chemotherapy drugs, so that to explore an effective way to clear MRD starting from the hematopoietic microenvironment is a new entry point.
The group's past research has shown that: under the condition of the appropriate combination of cytokines, human umbilical cord blood-derived stromal cells (hUCBDSCs ) can be successfully cultivated and amplified; after the amplification, the cells composition and immunophenotype of the hUCBDSCs is similar to that of hBMSCs, which can secrete a variety of cytokines and carries the similar material basis for supporting and reconstructing the hematopoietic function with hBMSCs, so that it may be called hematopoietic inductive microenvironment from human umbilical cord blood-derived stromal cells (hUCBDSC-HIM).
The co-culture of human acute T Lymphocytic leukemia cell line Jurkat cells respectively with hUCBDSC-HIM and hBMSC-HIM has found: hUCBDSC-HIM has a stronger inhibition and the phenomenon of apoptosis promotion to the Jurkat cells compared with hBMSC-HIM.
Based on this point, the experiment will study the influences of hemopoietic microenvironment simulated by stromal cells from different sources to the biological characteristics of residual drug resistant Jurkat cells, such as generation cycle disposition, proliferation, differentiation, apoptosis and drug sensitivity systematically and in-depth, and carry on the preliminary study of its mechanisms.
Contents and methods:
1. Influence of microenvironment of normal and acute lymphoblastic leukemia bone marrow stromal cells to Jurkat cells’biological characteristics respectively
Groups: Jurkat suspended culture group; Jurkat/nomal hBMSCs co-culture group; Jurkat leukemic hBMSCs co-culture group.
The spatial relationship between the Jurkat cells and stromal cells was observed under the inverted microscope and scanning electron microscope. MTT method for the detection of the adhesion rate of Jurkat cell; Transwell method for the detection of Jurkat cell migration and invasion function changes; CCK-8 method, Real-time Q-PCR method, Western blot method for the detection of Jurkat cell proliferation characteristics change; flow cytometry for the detection of Jurkat cell cycle and DNR dose perturbation changes; flow cytometry method, transmission electron microscopy, Real-time Q-PCR method, Western blot method for the detection of Jurkat cells apoptosis; NBT reduction method for the detection of Jurkat cell differentiation level changes.
2. Preliminary study on the culture and its drug resistance mechanism of residual drug resistance Jurkat cell
We use the isolated culture of BMSCs in patients with primary acute lymphoblastic leukemia in vitro to simulate the bone marrow hematopoietic microenvironment. In the long process of co-culture with Jurkat cells, we choose the culture medium containing 50ng/ml DNR. With the culture time extended, the vast majority of Jurkat cells are floating dead. A very small number of residual Jurkat cells recover and got the drug resistant ability that can survive and proliferate in the culture medium with 50ng/ml final concentration of DNR, these cells are recorded as Jurkat / DNR cells. We use CCK-8 method to detect the proliferation characteristics and the resistance coefficient of a wide range of chemotherapy drug of Jurkat and Jurkat / DNR cell; flow cytometry method for the detection of generation cycle and the dose perturbation of the DNR for Jurkat and Jurkat / DNR cell; through the detection of Caspase - Glo 3 / 7 activity, Real-time Q-PCR method, Western blot method for the detection of apoptosis-related indicators of Jurkat and Jurkat / DNR cell; Real-time Q-PCR method for multidrug resistance-associated gene of Jurkat and Jurkat / DNR cell : expression of MDR1 and MRP mRNA.
3. The influences of hUCBDSCs to the residual drug resistant Jurkat cells
Groups: Jurkat / DNR cell suspension culture group; Jurkat / DNR cells / normal hBMSCs co-culture group; Jurkat / DNR cells / hUCBDSCs co-culture group; Jurkat / DNR cells / leukemia hBMSCs co-culture group (set only sensitive to IDA). Collect the Jurkat / DNR cells after suspension culture and co-cultured 10 days to detect their changes in proliferation, generation cycle disposition, apoptosis, differentiation, drug sensitivity of the IDA and the expression level of multidrug resistance gene MRD1 mRNA.
Results:
1. Inverted microscope and scanning electron microscopy: With the culture time extended, the normal hBMSCs and leukemia hBMSCs adhere to, combine with, and wrap the Jurkat cells, and they have the shielding function to the co-cultured Jurkat cells, and they will affect its biological characteristics in a certain extent.
1.1 Adhesion and migration function: the adhesion and chemotaxis function to Jurkat cells of leukemia hBMSCs is stronger than normal hBMSCs.
1.2 Inhibition of proliferation: Inhibition of of Jurkat cell’s proliferation by normal hBMSCs is stronger than leukemia hBMSCs.
1.3 Generation cycle disposition: compared with the Jurkat cells in suspension culture, the Jurkat cells proportion in G0/G1 + G2 / M phase of the hBMSCs leukemia cells co-cultured group has significantly increased; the proportion of cells in S + G2 / M phase in the normal hBMSCs co-culture group has significantly increased.
1.4 For the Jurkat cell apoptosis: hBMSCs leukemia inhibit Jurkat cell apoptosis, while normal hBMSCs promote Jurkat cell apoptosis.
1.5 For the Jurkat cell differentiation: the promote-differentiation function of hBMSCs leukemia to the Jurkat cells co-cultured is weaker than normal hBMSCs.
1.6 For the DNR dose changes: the dose of Jurkat cells in the co-cultured prostate group was significantly lower than the suspension culture group, of which Jurkat cells in the hBMSCs leukemia co-culture group is the lowest.
2. Jurkat cells peg-and-socket joined in matrix cell gradually get the drug resistance continuously stimulated by the DNR, left the DNR resistance of Jurkat cells. Compared with Jurkat cells, Jurkat / DNR cells has the following characteristics:
2.1 Proliferation curve: the proliferation speed of Jurkat / DNR cells significantly slow down than Jurkat cells.
2.2 Generation cycle disposition: the proportion of cells in G0/G1 phase increased; the proportion of different diploid cells increased.
2.3 Apoptosis rate: the occurrence of spontaneous apoptosis of cells significantly reduced.
2.4 DNR dosage: the average drug concentration in a single Jurkat / DNR cell markedly reduced.
2.5 Multi-drug resistance to a wide range of chemotherapy drug.
2.6 Multi-drug resistance-associated gene expression: in the Jurkat / DNR cells, we can detect the expression of MDR1 mRNA; its expression level of MRP mRNA increased.
3. hUCBDSCs to the residual drug resistanct Jurkat cells
3.1 Proliferation curve: inhibition of cell proliferation of hUCBDSCs to Jurkat / DNR cells is stronger than normal hBMSCs.
3.2 Generation cycle disposition: hBMSCs and normal hUCBDSCs can both promote Jurkat / DNR cells to the generation cycle, the proportion of cells in G0/G1 phase of hUCBDSCs co-culture group is the lowest; the proportion of different diploid cells in Jurkat / DNR cells in the co-culture group significantly reduce.
3.3 To the cell apoptosis and differentiation: the promotion of hUCBDSCs to the spontaneous apoptosis and differentiation of co-cultured Jurkat / DNR cell is stronger than normal hBMSCs.
3.4 Sensitivity to IDA: hUCBDSCs and hBMSCs can increase the IDA sensitivity of Jurkat / DNR, and hUCBDSCs is superior to hBMSCs.
3.5 mRNA expression in MDR1: the expression of MDR1 mRNA level in co-culture group of Jurkat / DNR cell is lower than the suspension culture group, of which the expression level of the hUCBDSCs co-culture group is the lowest.
Conclusion:
1. Leukemia bone marrow stromal cells micro-environment can contribute to the shelter, growth and evasion of the anti-drug chemotherapy of leukemic cell; compared with Leukemia bone marrow stromal cells, normal bone marrow stromal cells has a stronger function in pro-apoptotic, differentiation and inhibit proliferation to Jurkat cells . At the same time, it can increase the sensitivity of the DNR of Jurkat cells.
2. The hematopoietic micro-environment simulated by leukemia bone marrow stromal cells can induce Jurakt cells to form multi-drug resistance, which is an important reason for the formation of residual drug resistance, suggesting that it is possible to inhibit the formation of residual leukemia and reduced recurrence by transforming the hematopoietic microenvironment in patients with leukemia.
3. Bone marrow-derived stromal cells and cord blood-derived stromal cells can both transform the hematopoietic microenvironment of leukemia, and reverse the drug resistance of Jurkat / DNR cells in a certain extent.
4. Cord blood-derived stromal cells have a stronger effect in the transformation of hematopoietic microenvironment of leukemia and the reverse of Jurkat / DNR cell drug resistance.
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