摘要
小鼠骨髓间充质干细胞对白血病细胞增殖的影响及机制探讨研究目的异基因造血干细胞移植是目前能够治愈白血病的一个重要途径,主要依靠移植前的强烈化疗和由供体免疫细胞介导的移植物抗白血病效应。然而,很大一部分患者在造血干细胞移植后由于白血病干／祖细胞的存在会导致疾病的复发,而增加放化疗的强度由于毒性反应的增加并不能提高生存率,因此,造血干细胞移植后的GVL效应尤为重要。骨髓间充质干细胞(mesenchymal stem cells,MSC)是具有高度自我更新能力和多向分化潜能的成体干细胞,大量的体内外实验及临床研究证实骨髓间充质干细胞有一定的免疫抑制作用,能够减轻异基因移植后的移植物抗宿主病。但是,MSC对白血病细胞的增殖究竟是抑制作用还是促进其增殖,或者对其无明显影响?对临床应用MSC与造血干细胞共移植治疗白血病有重要意义,目前这方面的研究较少,尚无定论。
针对以上现状,本课题拟解决以下三个问题：1、通过静脉输注少量A20细胞,建立微小残留白血病的异基因骨髓移植模型,研究静脉输注异基因骨髓MSC对异基因骨髓移植模型A20细胞增殖的影响；2、通过体外间充质干细胞与不同来源的白血病细胞共培养,观察间充质干细胞对白血病细胞增殖及其生物学特性的影响；3、建立A20皮下肿瘤模型,研究静脉输注和局部注射异基因骨髓源MSC对A20皮下肿瘤增殖的影响。从而为临床上造血干细胞和间充质干细胞联合移植治疗恶性血液病提供一定的实验依据。
第一部分小鼠骨髓间充质干细胞抑制异基因骨髓移植模型中A20细胞的增殖
研究方法1、建立以C57BL/6小鼠为供体、致死剂量照射的BABL/c小鼠为受体的A20淋巴瘤／白血病异基因小鼠骨髓移植模型,按照移植当天分别输注的细胞种类骨髓细胞1×107/只、MSC 5×105/只及A20细胞1×104/只,共分成5组：PBS组(n=10)、单纯骨髓组(n=10)、MSC-骨髓组(n=10)、单纯A20白血病组(n=17)、MSC-A20白血病组(n=17)；2、观察移植后小鼠的一般状态、体重变化、生存率及小鼠死亡原因,绘制体重曲线、生存曲线及两组小鼠肝脾淋巴瘤发生率；3、分别于移植后第7、14天流式细胞术检测外周血T细胞亚群；4、肝、脾、小肠、肺脏病理切片观察A20细胞的脏器浸润及脏器损伤情况；5、PHK26标记MSC,荧光显微镜观察MSC的脏器归巢情况。
结果1、异基因骨髓移植后28天MSC-A20组小鼠A20肝脾淋巴瘤发生率为58.82%,低于未输注MSC组(94.12%),统计学上有显著差异(P<0.05)；2、移植后第7天外周血T细胞亚群分析结果表明,MSC-A20组小鼠外周血中CD3+CD8+T细胞比例为(37.40±9.03)%,较未输注MSC组[(4.90±1.74)%]显著增加(P<0.01);MSC组CD3+CD4+T比例为(50.85±11.85)%,而CD4+CD25+T细胞比例则(1.88±0.43)%,显著低于未输注MSC组的(78.41±3.98)%和(4.73±1.67)%(P<0.05)。而移植后14天两组外周血T细胞亚群无显著差异；3、MSC-A20组小鼠的小肠、肺脏损伤情况较单纯A20组轻；4、MSC在移植后24小时能够归巢到脾脏和肾脏；移植后第7天能够归巢到脾脏、肝脏、肺和肾脏,可能具有向肿瘤部位及受损伤部位趋化的作用。
第二部分小鼠骨髓间充质干细胞体外对白血病细胞增殖及生物学特性的影响
研究方法1、复苏本实验室冻存的P10小鼠骨髓间充质干细胞,流式细胞术检测细胞免疫表型；培养扩增mMSC,传代至P10-15消化后应用；MSC重悬后加入丝裂霉素(25μg/ml,孵育20min)去除MSC的增殖能力(MMC-MSC), PBS洗涤3次,完全培养液重悬备用。按照2×103/孔,5×103/孔,2×104/孔,5×104/孔分别接种于96孔板(n=6),与FBL3、A20、P388细胞共培养(MSC与白血病细胞比例分别为1：10,1：4,1：1,1：0.4)。同时设置单纯A20、FBL3、P388对照组及单纯MMC-MSC组,共培养44小时后CCK-8法检测MSC对白血病细胞增殖的影响。2、MMC-MSC按照2×104/孔接种于96孔板,与A20、FBL3、P388以1：1的比例分别共培养24h、48h、72h, CCK-8法检测MSC抑制白血病细胞增殖的时间依赖性；3、MMC-MSC按照4×104/孔接种于Transwell板中,Transwell小室中加入同样数量的CFSE染色的A20细胞,共培养48小时后检测MSC在Transwell中对A20细胞增殖的影响；4、MMC-MSC按照1×105/孔接种于6孔板中,完全贴壁后加入同样数量的白血病细胞(A20、FBL3、P388),共培养72小时后,收集悬浮的白血病细胞,流式细胞术检测白血病细胞早期凋亡率及细胞周期；5、MMC-MSC按照4×104/孔接种于Transwell板中,Transwell小室中加入同样数量的A20细胞,共培养72小时后,流式细胞术检测Transwell中MSC对A20细胞周期及早期凋亡的影响；6、MMC-MSC按照2×104/孔接种于96孔板,A20细胞以1：1比例与其共培养,分别设立MSC+A20细胞共培养组,Akt inhibitor+A20组,及MSC+A20+Akt inhibitor组,检测MSC抑制A20细胞增殖与Akt激酶的关系；7、MMC-MSC以1：1比例与白血病细胞共培养于6孔板中,72小时后收集对照组和实验组的白血病细胞,Real-time PCR方法检测MSC对白血病细胞p21、caspase 3基因mRNA水平的影响；8、MMC-MSC与A20细胞以1：1比例共培养于6孔板中,分别于24h、48h、72h后收集上清,ELISA方法检测MSC对A20细胞分泌细胞因子IL-10、TGF-β、TNF-α和IFN-γ的影响；9、两者共培养48小时后,收集实验组和对照组的A20细胞,流式细胞术以CD19设门,检测MSC对A20细胞胞内IL-10的影响。
结果1、复苏及传代的MSC高表达CD29、CD44、Sca-1、MHC-1,中度表达CD13、CD90.2,不表达造血细胞表面标志CD117、CD45,不表达Flk-1、MHC-Ⅱ类抗原；且P15 MSC裸鼠体内无致瘤性；2、小鼠骨髓MSC在体外能够抑制不同来源的白血病细胞增殖,呈浓度依赖性,mMSC与A20、FBL3、P388共培养比例为1：0.4时,抑制效果最明显,与对照组相比,其相对增殖率分别为(78.32±9.16)%、(52.43±17.10)%、(58.47±5.68)%(P<0.01)；3、MSC对不同来源白血病细胞的增殖呈时间依赖性,随着共培养时间延长,MSC的抑制作用增强(P<0.05)；4、异基因小鼠骨髓MSC并非通过抑制Akt激酶而发挥抑制A20细胞增殖的作用(P<0.05)；5、MSC能够增加不同来源白血病细胞的早期凋亡率,与MSC共培养72小时后,A20、FBL3、P388细胞的早期凋亡率分别为(4.80±0.78)%、(12.88±2.78)%、(18.28±1.72)%,而对照组则分别为(1.06±0.21)%、(4.64±0.61)%、(7.70±0.61)%；6、MSC能够抑制不同来源白血病细胞的DNA合成,与MSC共培养72小时后,A20、FBL3、P388细胞的S期的比例分别为(45.77±1.56)%、(38.40±7.250%、(51.03±2.10)%,而对照组则分别为(62.93±1.20)%、(63.38±14.47)%、(56.27±0.55)%(P<0.05)；7、异基因小鼠骨髓MSC抑制A20细胞的增殖、阻滞细胞周期进展及促进A20细胞的早期凋亡作用需要两者直接接触；8、Real-time PCR检测结果表明小鼠骨髓源MSC能够增加不同来源白血病细胞p21、caspase3基因mRNA水平的表达(P<0.05)；9、异基因骨髓MSC能够减少A20细胞上清中IL-10的浓度,并呈时间依赖性,MSC与A20细胞共培养24h、48h、72h后上清中IL-10浓度分别为(236.99±15.91) pg/ml、(318.76±15.91) pg/ml、(680.41±78.07) pg/ml,而单纯A20组培养24h、48h、72h后上清中的IL-10浓度分别为(301.47±30.31)pg/ml、(554.62±19.79) pg/ml、(1892.97±176.22) pg/ml; MSC对A20细胞上清中TGF-p、TNF-α和IFN-γ水平无显著影响；10、小鼠异基因骨髓源MSC体外能够显著增加A20细胞胞内IL-10,与MSC共培养48小时后,流式细胞术检测A20细胞胞内IL-10较对照组显著增加,为(4.64±0.89)%,而未共培养组的A20细胞的胞内IL-10仅为(1.74±0.59)%(P<0.01)。
第三部分小鼠骨髓间充质干细胞对A20皮下肿瘤增殖的影响
研究方法1、A20细胞以1×106/只接种于BABL/c小鼠或BABL/c裸鼠皮下,建立A20皮下移植瘤模型；根据MSC(5×105/只)与A20细胞同时皮下注射或者静脉注射于小鼠体内做如下分组：(MSC+A20)/SC组和MSC(IV)+A20(SC);而受体鼠则分别为BABL/c小鼠或BABL/c裸鼠；2、MSC局部注射的剂量降低为1×105/只,与A20细胞同时同部位注射于BABL/c小鼠或BABL/c裸鼠皮下；3、致瘤后检测指标：小鼠肿瘤发生时间、小鼠皮下肿瘤体积、小鼠肿瘤病理、肿瘤免疫组织化学PCNA、CD31及BABL/c小鼠致瘤后第7天外周血T细胞亚群。
结果1、大剂量MSC与A20细胞同部位皮下注射于有免疫力的BABL/c小鼠,能够促进A20肿瘤的增殖,致瘤24天后(MSC+A20)/SC组小鼠平均肿瘤体积达(1511.43±467.85)mm3,而未注射MSC组小鼠肿瘤体积仅为(615.36±201.22)mm3(P<0.05); (MSC+A20)/SC组小鼠致瘤后第13天100%发生皮下肿瘤,而单纯A20组小鼠仅有83%的小鼠发生皮下肿瘤；第7天检测外周血T细胞亚群两组无显著差异(P>0.05)；两组小鼠肿瘤免疫组化分析表明,(MSC+A20)/SC组小鼠PCNA、CD31表达略高于单纯A20组；2、大剂量MSC与A20细胞同部位皮下注射于BABL/c裸鼠,能够促进A20皮下瘤的增殖,致瘤后20天MSC组小鼠的皮下肿瘤体积达(1993.34±1009.89)mm3,而未注射MSC的裸鼠皮下肿瘤体积仅为(604.88±235.92)mm3(P<0.05)；致瘤后16天MSC组小鼠即100%发生皮下肿瘤,而未注射MSC的裸鼠皮下发生率仅为40%；两组小鼠肿瘤免疫组化分析表明,(MSC+A20)/SC组小鼠PCNA、D31表达略高于单纯A20组；3、静脉注射MSC对BABL/c小鼠以及BABL/c裸鼠皮下瘤的增殖动力学及发生率均无显著影响；对BABL/c小鼠致瘤后第7天外周血T细胞亚群亦无显著影响。4、无论在BABL/c小鼠还是BABL/c裸鼠,小剂量MSC局部注射于A20肿瘤部位对局部肿瘤的发生率及肿瘤体积无显著影响。结论1、体外复苏传代的P10 mMSC细胞表面标志表达及细胞形态跟冻存前相同,培养扩增的P15 mMSC裸鼠体内无致瘤性,生物学特性稳定；2、静脉输注MSC能够抑制异基因骨髓移植后4周A20小鼠肝脾淋巴瘤的发生率,主要是通过增加外周血中CD3+CD8+T细胞比例,减少CD3+CD4+T和CD4+CD25+T细胞比例而发挥作用；3、在异基因骨髓移植的A20白血病小鼠模型中,静脉输注小鼠异基因骨髓MSC能够归巢到脾脏、肝脏、肺及肾脏,可能发挥修复照射损伤的器官及局部抗肿瘤的作用；4、小鼠骨髓源MSC体外能够抑制不同来源的白血病细胞增殖,呈浓度和时间依赖性；5、小鼠骨髓源MSC体外能够促进不同来源白血病细胞的早期凋亡、抑制白血病细胞DNA合成；6、MSC需要与白血病细胞直接接触才能发挥其增殖抑制、促凋亡及周期阻滞作用；7、小鼠异基因骨髓源MSC体外能够抑制A20细胞分泌IL-10;8、无论在有免疫力的BABL/c小鼠还是T细胞免疫缺陷的BABL/c裸鼠,大剂量异基因小鼠骨髓MSC局部注射于A20皮下肿瘤部位均能够促进肿瘤增殖,而静脉注射MSC则对皮下肿瘤增殖无显著影响,表明MSC静脉注射具有一定的安全性。
Objective Allogeneic hematopoietic stem cell transplantation is the preferred treatment for many hematologic malignancies,which mostly depend on the intensive chemotherapy and graft versus leukemia effects.However,many leukemias will relapse in some patients due to the existence of leukemia stem cells.With the increasing of the radiotherapy and chemotherapy intensities,the patients'survival rates can't be boosted.Accordingly,the graft versus leukemia effects play an important role in hematopoietic stem cell transplantations.
Bone marrow derived mesenchymal stem cells are known for their characteristics of being postnatal stem cells,having the capacity of self-renewal and differentiation into the mesenchymal lineage.Many experiments prove that they have immunosuppressive functions in vivo and in vitro,and they can alleviate GVHD.But the effects of mesenchymal stem cells on leukemia cells have not been known,which will play an important role in the clinical application of the transplantations of both MSC and hematopoietic stem cells.
Based on such a status quo,our experiment will solve three problems:1.we will detect the effects of mMSC on the proliferation and biology of leukemia cells through the experiment ex vivo;2. Intravenously infusion of a small amount of A20 cells,to establish a minimal residual leukemia in allogeneic bone marrow transplantation model, we study infusion of allogeneic bone marrow-derived MSC on the A20 cells proliferation in allo-BMT model;3.To establishment of a BABL/c mouse subcutaneous A20 tumor model, we study intravenously infusion of or local injection of allogeneic bone marrow-derived MSC on the effect of proliferation of A20 subcutaneous tumor.Then we can provide some experimental basis for clinical hematopoietic stem cells and mesenchymal stem cells transplantation of treating malignant hematological diseases.
Part one mMSC can inhibit the proliferation of A20 cells in allogeneic bone marrow model
Methods 1.Allogeneic BMT model was established as follows:lethally irradiated BABL/c recipients received allogeneic BM cells of C57BL/6 origin.The mice were divided into 5 groups according to the infusion cell types:①PBS group(n=10):PBS 0.6ml;②BM group(n=10):1×107 BM cells per mice;③MSC-BM group(n=10):1×107 BM cells+5×105 MSCs;④A20 group(n=17):1×107 BM cells+1×104 A20 cells;⑤MSC-A20 group(n=17):1×107 BM cells+1×104 A20 cells+5×105 MSCs.2.Compare the weight curve,survival rate,and tumor incidence,and analyze the T cells subset in peripheral blood at day 7 and day 14 after allogeneic BMT.3. Liver,spleen,small intestine and lung biopsy were stained with Hematoxylin-eosin,and compare the infiltration of A20 cells and the organ injuries.4.PKH26 labeled MSCs were transplanted into allo-BMT model to observe the distribution of mMSCs.
Results 1.The mean survival time in PBS group was 15 days,while 90% mice in BM group and 100% mice in BM-MSC group were survived over 29 days.2.The tumor incidence of MSC-A20 group at day 28 after allo-BMT was 58.28%,which was lower than that of A20 group(94.12%)(P<0.05);3.Allogeneic MSCs infusion could increase the CD3+CD8+T cell subset in peripheral blood to (37.40±9.03)%,which was significantly higher than A20 group(4.90±1.74)% at day 7 after BMT(P<0.01);The percent of CD3+CD4+T cell was (50.85±11.85)%,and CD4+CD25+T cell was (1.88±0.43)% in MSC-A20 group,which were both lower than in A20 group(P<0.05).But MSCs infusion had no influence on the percentage of T cell subset at day 14 after allogeneic BMTAmMSCs were homing to the spleen,liver,lung and kidney,perhaps related with repairment the injury organs.
Part two The effects of mMSC on the proliferation and biology of leukemia cells in vitro
Methods 1.Reanimate the freezing and preserving mesenchymal stem cells of passage ten to fifteen,and the cell's phenotypes were demonstrated.MSCs were inactivated in MSC medium supplemented with mitomycin(25μg/ml) for 20 minutes at 37℃in advance, then were washed with PBS three times.They were inoculated in 96-well flat-bottomed microplates in different quantities(i.e.2×103、5×103、2×104、5×104/well).2×104 leukemia cells (A20,FBL3,P388)were cultured with or without a increasing number of MMC-treated MSCs in 96-well plates for 48h(n=6).Proliferation was assessed by CCK-8 assay during the final of 4h of culture.2.Time-dependent analysis:2×104 leukemia cells were cultured in 96-well plates for different time(24h,48h,72h) in the presence of 2x104 MMC-treated MSCs,and proliferations of leukemia cells were analyzed by CCK-8 assay.3.The proliferation rate of A20 cells was evaluated in different groups for 48h. Control group:A20 cells only;Group1:A20+MMC-MSCs(1:1);Group2:A20+Akt inhibitor(5μM);Group3:A20 +MSCs(1:1)+Akt inhibitor(5μM);Group4:A20+transwell-seperated MSCs.The results are shown as percentage of cell proliferation in comparison with control leukemia cells proliferation.4. 1×105 leukemia cells were cultured for 72h alone or in presence of MMC-treated MSCs at ratio 1:1 (MSCs:Leukemia cells).Leukemia cells were harvested and analyzed for the percent of early apoptosis and cell cycle distribution.5. 1×105A20 cells were cultured as a ratio of 1:1 physically separated from MMC-MSCs using a transwell-system for 72h,then A20 cells were harvested and analyzed for the percent of early apoptosis and cell cycle.6.After cultured with or without MMC-MSCs for 72h,A20 cells of the two groups were harvested and extracted for total RNA,then was analyzed for p21 and caspase 3 gene expression with Real time PCR.7. 1×105 A20 cells were cultured with or without MMC-treated MSCs in 6-well plates at ratio 1:1 for 24h,48h,or 72h,respectively (n=5).Then the supernatants were harvested, and cytokines'levels (IL-10,TGF-β,TNF-a and IFN-y) in cultural supernatants of different time were measured by ELISA arrays.8.Expression of A20 cells from cultures with or without MMC-treated MSCs for 48h using IL-10 staining on CD19-gated cells in flow cytometry.
Results 1.The cells population consisted of spindle-and star-shaped cells.Flow-cytometric analysis showed that they were high positive for CD29、CD44、Scal-1、MHC-I, moderate positive for CD31、CD90.2 and negative for CD117、CD45、Flk-1、MHC-II.MSCs were of no tumorigenicity in BABL/c nude mice.2. MSCs exhibited a number-dependent growth inhibitory effects on different hematopoietc original leukemia cells.When the ratio of mMSC and leukemia cells was 1:0.4,the inhibition effect was most obvious.Compared with the control groups,the relative proliferation rate of A20,FBL3,and P388 cells in cocultural groups were respectively (78.32±9.16)%,(52.43±17.10)%, (58.47±5.68)% (P<0.01).3.The inhibitory effect of mMSC on different leukemia cells was time-dependent when seeded at a ratio 1:1(P<0.05);4.MSCs can not inhibit the activation of the Akt protein kinase;5.Apoptosis of leukemia cells was dramatically increased after coclutured with MMC-MSCs for 72h,the early apoptosis rates of A20,FBL3 and P388 cells were (4.80±0.78)%,(12.88±2.78)%,and (18.28±1.72)% respectively,while the rates of controls were (1.06±0.21)%,(4.64±0.61)% and (7.70±0.61)% respectively.6.When cocultured with MMC-MSCs for 72h,the DNA synthesis of leukemia cells were dramatically decreased.The perecent of A20,FBL3 and P388 cells in S phase were (45.77±1.56)%,(38.40±7.25)% and (51.03±2.10)% respectively,while the rates of control were (62.93±1.20)%,(63.38±14.47)% and (56.27±0.55)%(P<0.05);7.The inhibitoy effect on the proliferation of leukemia cells, the arrest effect on the leukemia cell cycle and the promoting early apoptosis effect require MSCs to make direct cell-cell contact;8.The results of Real-time PCR demonstrated that after coclutured for 72h,MSCs could upregulated mRNA expression of p21 and caspase 3 in leukemia cells significantly(P<0.05);9.ELISA assay showed that MSCs could decrease the level of IL-10 in supernantant and was time-dependent.The IL-10 concentrations of coculture supernatants of 24h,48h,and 72h were (236.99±15.91)pg/ml, (318.76±15.91) pg/ml, and(680.41±78.07)pg/ml,while the controls'were (301.47±30.31)pg/ml,(554.62±19.79) pg/ml,and (1892.97±176.22)pg/ml respectively; 10.After cocultured with mMSC for 48h,the intracellular IL-10 of A20 cells was significantly increased to (4.64±0.89)%,while the control's was (1.74±0.59)%(P<0.01).
Part three The effect of mMSCs on the proliferation of A20 subcutanous tumor Methods 1.MSCs were either coimplanted as a mix(5x105MSC cells) with A20 cells(1×106) subcutaneously or injected alone via the tail vein.Groups were divided as follows:①(MSC+A20)/SC:MSCs and A20 cells were coimplanted subcutaneously.②MSC(IV)+A20(SC):MSCs were injected intravenously and A20 cells were implanted subcutaneously.③A20 alone:A20 cells were implanted subcutaneously without MSCs.Recipient mices were BABL/c mice or BALB/c nude mice.2.The tumor incidence,tumor mean volumes,tumor histology,immunohistochemitry of PCNA and CD31 of tumors and T cell subset of day 7 in peripheral blood were detected after implantation.
Results 1.Coimplantion with MSCs and A20 cells could promote the proliferation of A20 cells in BALB/c mice.The mean volume of (MSC+A20)/SC in BALB/c mice at day 24 after implantation was (1511.43±467.85)mm3,while that in A20 alone group was (615.36±201.22)mm3(P<0.05);Tumor incidence reached 100% at day 13 after MSCs and A20 cells were coimplanted subcutaneously,while that in A20 alone group was only 83%;The T cell subset at day 7 in peripheral blood were of no significant difference(P>0.05);Immunohistochemitry analysis showed that PCNA and CD31 expression in (MSC+A20)/SC was higher than A20 alone group;2.Coimplantion with MSCs and A20 cells could promote the proliferation of A20 cells in BALB/c nude mice.The mean volume of (MSC+A20)/SC in BALB/c mice at day 20 after implantation was (1993.34±1009.89)mm3, while that in A20 alone group was (604.88±235.92)mm3 (P<0.05);Tumor incidence reached 100% at day 16 after MSCs and A20 cells were coimplanted subcutaneously,while that in A20 alone group was only 40%; Immunohistochemitry analysis showed that PCNA and CD31 expression in (MSC+A20)/SC was higher than A20 alone group;3.There were no significant difference of mean tumor volumes and tumor incidence when MSCs were injected intravenously while A20 cells were implanted subcutaneously both in BABL/c mice and in BALB/c nude mice;Allogeneic MSCs had no effect on the T cell subset at day 7 after implantation when they were injected intravenously.
Conclusions 1.When MSCs were injected via vein,they could decrease the tumor incidence of A20 tumor in allogeneic BMT model through increasing the percent of CD3+CD8+T cells and decreasing the percent of CD3+CD4+T and CD4+CD25+T cells;2.MSCs could be homing to the spleen,liver,lung and kidney,perhaps related with repairing the injury organs.3. Mouse bone marrow-derived MSCs could inhibit the proliferation of different leukemia cells and was concentration-dependent and time-dependent;4. Mouse bone marrow-derived MSCs can promote the early apoptosis of different leukemia cells and arrest the cell cycle of leukemia cells;5.The inhibitoy effect on the proliferation of leukemia cells, the arrest effect on the leukemia cell cycle and the promoting early apoptosis effect require MSCs to make direct cell-cell contact;6. MSCs could inhibit the level of IL-10 in A20 culture supernantant and was time-dependent; 7.MSCs could increase the tumor incidence and the mean tumor volumes when they were coimplanted with A20 cells subcutaneously both in BALB/c mice and in BALB/c nude mice,which were not irrelevant with T cell immunity;There was no effect on the proliferation of A20 subcutous tumors when MSCs were injected intravenously.
引文
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