多药耐药基因转染人胎盘源性间充质干细胞及其对化疗保护作用的实验研究
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摘要
为探讨多药耐药基因(mdr1)导入人胎盘源性间充质干细胞(MSCs)能否提高其对化疗药物的耐受性,我们采用灌流法联合密度梯度离心法自胎盘组织中分离扩增MSCs,脂质体转染法将携带有mdr1基因的逆转录病毒载体导入293T包装细胞,获得的病毒上清重复感染MSCs,通过RT-PCR、流式细胞术及免疫荧光染色检测mdr1基因在MSCs基因组中的整合和表达,同时对转染细胞的生物学特性及多向分化潜能加以测定;罗丹明排泌试验检测基因编码产物P-g170的功能活性,MTT法测定转染MSCs对多种化疗药物耐受性。在此基础上,采用γ-Co60照射法建立裸鼠骨髓造血功能损伤模型,研究转染MSCs经静脉回输后对紫杉醇化疗的保护作用。结果表明,mdr1基因可于MSCs中稳定转染和表达,转染后MSCs仍符合干细胞的生物学特性。基因转染后细胞可表达有功能活性的P-g170,且表现出典型的多药耐药表型,mdr1-MSCs体内移植可对裸鼠外周血和骨髓细胞的生成起化疗保护作用。结果提示,由逆转录病毒载体介导mdr1基因转染人胎盘源性MSCs可获得高效、稳定的功能性蛋白表达,此转染行为对于维持MSCs的干细胞特性是安全可行的,通过转染MSCs的体内移植能够增强机体对化疗的耐受程度,从而有望为提高大剂量化疗在肿瘤治疗中的应用提供可靠保证。
Most of gynecologic malignances are sensitive to chemotherapy. But the bone marrow (BM) toxicity and other side effects might indirectly impair the efficiency of consolidation chemotherapy. Currently, the emphasis is to study how to increase the resistance of normal cells to drugs and how to minimize its side effects.
Studies indicated that the excess expression of mdr1 gene was the main reason of multidrug resistance (MDR). The gene was not only the resistant gene to malignant cells, but also the protective gene to normal cells. The mdr1 gene product, P-glycoprotein 170(P-g170), is a transmembrane efflux pump that shunts a variety of naturally chemotherapeutic agents out of cells..
Mesenchymal stem cells (MSCs) have been paid more attention as a kind of stem cells derived multiple tissues. It has been approved marrow MSCs have the capability to support the expansion of hematopoietic stem cell(sHSCs)through expressing hematopoietic cytokines and reconstructing hematopoietic microenviroment. And the adhesion molecule and extracellular matrix expressed by MSCs also play a role in the homing of HSCs. In the same time, MSCs have the capability to differentiate to stroma cells under certain induced conditions, which might be supplementary for marrow haemopoiesis.
To explore whether the transfection of retrovirus vector-mediated mdr1 gene into MSCs could increase resistance of MSCs to cytotoxic drugs, human placenta MSCs were isolated and expanded by a Percoll density gradient. The cells remained fibroblastlike morphological features. The retrovirus vector carrying mdr1 and green fluorescent protein (GFP)genes was transferred to the packaging cells, 293T by lipofectamine mediated gene transfer. Viral titer was made into 5×106CFU·mL-1 by high speed centrifugation, and the transduction efficiency was above 90%. The expression of GFP gene was observed through laser confocal microscopy. MSCs were infected with the concentrated virus. After screening with puromycin, flow cytometric (FCM) analysis showed the stable transduction efficiency was 80.3%. Total RNA extracted from the mdr1-MSCs was detected by RT-PCR and a positive strap expected length 310bp was expanded, which confirmed that mdr1 gene had been integrated into the genome of MSCs. The P-g170 expression detected by immunofluorescence staining and FCM showed that there was increased expression of P-g170 on the surface of the transfected cells, and there were over 30% cells expressing the protein. Above results provided methodology foundation for in-depth study.
Then we observed and detected the characteristics of mdr1-MSCs. FCM analysis was adopted to determine the immunophenotypes. The result showed the cells were positive for CD29, CD44, the stem cells antigen CD73 (SH3, SH4) and negative for the markers of the hematopoietic lineage, including the CD34, the leukocyte common antigen CD45, and HLA-DR (MHC-II molecule). The growth kinetics was measured by MTT and the mean cumulative time of population doublings was 23.9h. The DNA content was analyzed to analyse cellular cycle, according with the characterization of stem cells, mostly cells were at quiescent phase (G0/G1 phase), while very few cells was at fissional phase (G2/M phase). The ultrastructures of MSCs demonstrated the features of poorly differentiated stem cells, including irregular cell shape, dumpy microvilli on the surface of cells, and well-distributed intranuclear chromatin. In addition, there were plentiful mitochondria and lightly broaden rough endoplasmic reticulum in the cytoplasm of the cells., suggesting they had doughty capacity of protein synthesis and secretion. The differentiation potential was tested by culturing transfected cells under conditions that favored adipogenic and osteogenic differentiation. The results confirmed that the mdr1-MSCs still retained their multiple differentiation.
The rhodamin 123 (Rh-123) efflux assay was performed to determine the function and activity of P-g170 expressed by mdr1-MSCs. After the transduced and non-transduced MSCs incubated with 1.5μg·mL-1 Rh-123 for 15min and 30min, red fluorescence was stired up and light intensity reinforced with the prolongation of incubation time. At the identical incubation time, the intensity was significantly weaker in transduced group than control group. FCM analysis showed that the mean fluorescence intensity in the transduced group was lower than that in non-transduced group after cells incubated with Rh-123 for 15min, 30min and 60min, which indicated that MSCs transduced by mdr1 gene had the enhanced capacity to extrude Rh-123 and expressed active P-g170. The resistance of mdr1-MSCs to cytotoxic drugs detected by MTT demonstrated there was a significant increase in the resistance of the transduced MSCs to chemotherapeutics including paclitaxel, vincristine, daunomycin and carboplatin, and the resistance factor increased respectively 4.2, 8.0, 3.5 and 1.8 times compared with the non-transduced MSCs. But there was no marked difference in the resistance to methotrexate and cyclophosphamide between the two groups of MSCs, suggesting that MSCs from human placenta might possess multidrug resistance phaenotype by mdr1 gene transfer.
Based on above studies, we established myelosuppression model byγ-Co60 radiation. The mdr1-MSCs or MSCs were infused via tail veins into model mice. Fourteen days after transplantation, mice received intraperitoneal (i.p.) injections of paclitaxel or phosphate-buffered saline (PBS) lasting for a period of 2 weeks. The accumulated dose was up to 120mg/kg body weight. The survival of these nude mice was observed everyday. Peripheral blood (PB) was collected from tail veins and before, during and after treatment with paclitaxel or PBS, and PB cell counts for white blood cells (WBC), as well as red blood cells (RBC), platelets (PLT) and values for hemoglobin (Hb) were determined using an antomatic analyzer. Nude mice were put to death at day 37 after transplantation, and the rinse solution of mice cavitas medullaris was collected for counting BM nucleated cells. A Percoll density gradient was used to isolate mononuclear cells from BM nucleated cells. The cell smears were observed the GFP expression by fluorescence microscope, and FCM analysis was used to count the GFP-labeling cells. Results showed that the survival rate of animals increased for paclitaxel treated mdr1-MSCs mice, which suggested the transplantation with mdr1-MSCs might improve the resistance to the drug- paclitaxel and increase the vitality. One weak after paclitaxel treatment, significantly decreased PB cells counts were observed, and the counts of RBC, PLT and values of Hb in paclitaxel treated MSCs-mice were all lower than paclitaxel treated mdr1-MSCs-mice. This indicated that the transplanted mdr1-MSCs might promote marrow haematogenesis by secreting certain factors related with haematogenesis, thus weaken the depressant effect of chemotherapy. Because of short life period of WBC and its high sensitivity to the lethal effect of drugs, there was no significant difference in the decreased WBC counts of the two groups. At the ending of chemotherapy, the assay of PB cells counts showed the counts of WBC, RBC and values of Hb were all higher in mdr1-MSCs mice than MSCs mice, especially, mdr1-MSCs mice showed a tendency for an increase rather than a decrease of cell numbers three days after the ending of chemotherapy. At the ten days after the ending of chemotherapy, the counts of RBC and the values of Hb had increased up to the level of PBS treated md1-MSCs mice. In addition, there was marked difference in the counts of BM nucleated cells of the two groups. Above results illustrated the transgenic MSCs might confer chemoprotection for the generation of PB and BM cells. The smears of BM mononuclear cells and FCM assay confirmed that the mdr1-MSCs derived from human placenta could be implanted into bone marrow of the animals, and keep uniform and regular shape. The percentage of implanted GFP-labeling MSCs in BM mononuclear cells was up to 18.33%. This indicated the transplanted MSCs could migrate to and locate at the damaged marrow, and keep well after implantation.
In conclusion, efficient and stable expression of mdr1 gene could be obtained by retroviral-mediated transfection, which was safe and feasible for keeping the biological characteristics of MSCs. The transgenic MSCs might reinforce the resistance to chemotherapy after cells transplantation. It is expected to provide a reliable warranty for the application of high-dose anticancer agents.
Most of gynecologic malignances are sensitive to chemotherapy. But the bone marrow (BM) toxicity and other side effects might indirectly impair the efficiency of consolidation chemotherapy. Currently, the emphasis is to study how to increase the resistance of normal cells to drugs and how to minimize its side effects.
Studies indicated that the excess expression of mdr1 gene was the main reason of multidrug resistance (MDR). The gene was not only the resistant gene to malignant cells, but also the protective gene to normal cells. The mdr1 gene product, P-glycoprotein 170(P-g170), is a transmembrane efflux pump that shunts a variety of naturally chemotherapeutic agents out of cells..
Mesenchymal stem cells (MSCs) have been paid more attention as a kind of stem cells derived multiple tissues. It has been approved marrow MSCs have the capability to support the expansion of hematopoietic stem cell(sHSCs)through expressing hematopoietic cytokines and reconstructing hematopoietic microenviroment. And the adhesion molecule and extracellular matrix expressed by MSCs also play a role in the homing of HSCs. In the same time, MSCs have the capability to differentiate to stroma cells under certain induced conditions, which might be supplementary for marrow haemopoiesis.
To explore whether the transfection of retrovirus vector-mediated mdr1 gene into MSCs could increase resistance of MSCs to cytotoxic drugs, human placenta MSCs were isolated and expanded by a Percoll density gradient. The cells remained fibroblastlike morphological features. The retrovirus vector carrying mdr1 and green fluorescent protein (GFP)genes was transferred to the packaging cells, 293T by lipofectamine mediated gene transfer. Viral titer was made into 5×106CFU·mL-1 by high speed centrifugation, and the transduction efficiency was above 90%. The expression of GFP gene was observed through laser confocal microscopy. MSCs were infected with the concentrated virus. After screening with puromycin, flow cytometric (FCM) analysis showed the stable transduction efficiency was 80.3%. Total RNA extracted from the mdr1-MSCs was detected by RT-PCR and a positive strap expected length 310bp was expanded, which confirmed that mdr1 gene had been integrated into the genome of MSCs. The P-g170 expression detected by immunofluorescence staining and FCM showed that there was increased expression of P-g170 on the surface of the transfected cells, and there were over 30% cells expressing the protein. Above results provided methodology foundation for in-depth study.
Then we observed and detected the characteristics of mdr1-MSCs. FCM analysis was adopted to determine the immunophenotypes. The result showed the cells were positive for CD29, CD44, the stem cells antigen CD73 (SH3, SH4) and negative for the markers of the hematopoietic lineage, including the CD34, the leukocyte common antigen CD45, and HLA-DR (MHC-II molecule). The growth kinetics was measured by MTT and the mean cumulative time of population doublings was 23.9h. The DNA content was analyzed to analyse cellular cycle, according with the characterization of stem cells, mostly cells were at quiescent phase (G0/G1 phase), while very few cells was at fissional phase (G2/M phase). The ultrastructures of MSCs demonstrated the features of poorly differentiated stem cells, including irregular cell shape, dumpy microvilli on the surface of cells, and well-distributed intranuclear chromatin. In addition, there were plentiful mitochondria and lightly broaden rough endoplasmic reticulum in the cytoplasm of the cells., suggesting they had doughty capacity of protein synthesis and secretion. The differentiation potential was tested by culturing transfected cells under conditions that favored adipogenic and osteogenic differentiation. The results confirmed that the mdr1-MSCs still retained their multiple differentiation.
The rhodamin 123 (Rh-123) efflux assay was performed to determine the function and activity of P-g170 expressed by mdr1-MSCs. After the transduced and non-transduced MSCs incubated with 1.5μg·mL-1 Rh-123 for 15min and 30min, red fluorescence was stired up and light intensity reinforced with the prolongation of incubation time. At the identical incubation time, the intensity was significantly weaker in transduced group than control group. FCM analysis showed that the mean fluorescence intensity in the transduced group was lower than that in non-transduced group after cells incubated with Rh-123 for 15min, 30min and 60min, which indicated that MSCs transduced by mdr1 gene had the enhanced capacity to extrude Rh-123 and expressed active P-g170. The resistance of mdr1-MSCs to cytotoxic drugs detected by MTT demonstrated there was a significant increase in the resistance of the transduced MSCs to chemotherapeutics including paclitaxel, vincristine, daunomycin and carboplatin, and the resistance factor increased respectively 4.2, 8.0, 3.5 and 1.8 times compared with the non-transduced MSCs. But there was no marked difference in the resistance to methotrexate and cyclophosphamide between the two groups of MSCs, suggesting that MSCs from human placenta might possess multidrug resistance phaenotype by mdr1 gene transfer.
Based on above studies, we established myelosuppression model byγ-Co60 radiation. The mdr1-MSCs or MSCs were infused via tail veins into model mice. Fourteen days after transplantation, mice received intraperitoneal (i.p.) injections of paclitaxel or phosphate-buffered saline (PBS) lasting for a period of 2 weeks. The accumulated dose was up to 120mg/kg body weight. The survival of these nude mice was observed everyday. Peripheral blood (PB) was collected from tail veins and before, during and after treatment with paclitaxel or PBS, and PB cell counts for white blood cells (WBC), as well as red blood cells (RBC), platelets (PLT) and values for hemoglobin (Hb) were determined using an antomatic analyzer. Nude mice were put to death at day 37 after transplantation, and the rinse solution of mice cavitas medullaris was collected for counting BM nucleated cells. A Percoll density gradient was used to isolate mononuclear cells from BM nucleated cells. The cell smears were observed the GFP expression by fluorescence microscope, and FCM analysis was used to count the GFP-labeling cells. Results showed that the survival rate of animals increased for paclitaxel treated mdr1-MSCs mice, which suggested the transplantation with mdr1-MSCs might improve the resistance to the drug- paclitaxel and increase the vitality. One weak after paclitaxel treatment, significantly decreased PB cells counts were observed, and the counts of RBC, PLT and values of Hb in paclitaxel treated MSCs-mice were all lower than paclitaxel treated mdr1-MSCs-mice. This indicated that the transplanted mdr1-MSCs might promote marrow haematogenesis by secreting certain factors related with haematogenesis, thus weaken the depressant effect of chemotherapy. Because of short life period of WBC and its high sensitivity to the lethal effect of drugs, there was no significant difference in the decreased WBC counts of the two groups. At the ending of chemotherapy, the assay of PB cells counts showed the counts of WBC, RBC and values of Hb were all higher in mdr1-MSCs mice than MSCs mice, especially, mdr1-MSCs mice showed a tendency for an increase rather than a decrease of cell numbers three days after the ending of chemotherapy. At the ten days after the ending of chemotherapy, the counts of RBC and the values of Hb had increased up to the level of PBS treated md1-MSCs mice. In addition, there was marked difference in the counts of BM nucleated cells of the two groups. Above results illustrated the transgenic MSCs might confer chemoprotection for the generation of PB and BM cells. The smears of BM mononuclear cells and FCM assay confirmed that the mdr1-MSCs derived from human placenta could be implanted into bone marrow of the animals, and keep uniform and regular shape. The percentage of implanted GFP-labeling MSCs in BM mononuclear cells was up to 18.33%. This indicated the transplanted MSCs could migrate to and locate at the damaged marrow, and keep well after implantation.
In conclusion, efficient and stable expression of mdr1 gene could be obtained by retroviral-mediated transfection, which was safe and feasible for keeping the biological characteristics of MSCs. The transgenic MSCs might reinforce the resistance to chemotherapy after cells transplantation. It is expected to provide a reliable warranty for the application of high-dose anticancer agents.
引文
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