慢病毒载体介导NK4基因修饰骨髓间充质干细胞靶向治疗胃癌的实验研究
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摘要
目的:
     1、建立人骨髓间充质干细胞(human bone-derived mesenchymal stemcells,hBMSCs)体外分离、培养、传代的方法;
     2、构建NK4和增强型绿色荧光蛋白(Enhanced green fluorescent protein, EGFP)融合基因的重组慢病毒表达载体;
     3、探讨hBMSCs在体内外对胃癌的趋向性;
     4、观察慢病毒介导NK4基因修饰的hBMSCs对胃癌的治疗效果及其相关抗肿瘤机制,期望为胃癌的基因治疗探索一条新途径。方法:
     1、hBMSCs分离培养及鉴定:采用全骨髓培养法分离培养,流式细胞仪检测CD34、CD44、CD45、CD105及骨诱导进行鉴定,成骨采用茜素红染色鉴定;
     2、采用聚合酶链反应(Polymerase chain reaction, PCR)从肝细胞生长因子(Hepatocyte Growth Factor, HGF) cDNA中克隆NK4基因,并经测序鉴定;以携带EGFP的pGC-FU慢病毒载体系统作为基因转导的媒介,采用In-Fusion技术构建NK4-EGFP融合基因的重组慢病毒表达载体,通过观察荧光及Western blot检测GFP蛋白表达;实时荧光定量PCR (Real-time quantitative PCR, RT Q-PCR)检测慢病毒滴度;
     3、将携带NK4-EGFP融合基因的慢病毒(Lenti-NK4)及仅携带EGFP的慢病毒(Lenti-GFP)转染hBMSCs,通过观察荧光及流式细胞仪检测转染率确定最佳感染复数(Multiplicity of infection, MOI);采用酶联免疫吸附试验(Enzyme linked immunosorbent assay, ELISA)及Western blot检测Lenti-NK4转染hBMSCs后NK4表达;
     4、建立裸鼠胃癌皮下移植瘤模型:采用胃癌细胞悬液皮下注射及瘤组织块接种两种方法;建立胃癌腹腔种植转移模型:采用胃癌细胞悬液及瘤组织块细胞悬液腹腔注射两种方法;
     5、hBMSCs对胃癌趋向性研究:(1)体外细胞迁移实验采用Transwell共培养体系,下室培养胃癌细胞株MKN45、胃上皮细胞株GES-1、同时设置空白对照组(培养基),24小时后上室分别加入hBMSCs、hFB,24小时后取出小室,结晶紫染色,显微镜下计数。(2)胃癌皮下移植瘤裸鼠成瘤后随机分为三组(每组5只),分别尾静脉注射hBMSCs-GFP、hFB-GFP、Lenti-GFP,7天后处死裸鼠,取瘤体、心、肝脏、脾脏、肺、肾脏,冰冻切片,荧光显微镜下观察瘤体及各脏器GFP表达情况。
     6、以hBMSCs为载体的NK4基因治疗对胃癌治疗效果:32只裸鼠成瘤后随机分为四组:hBMSCs-NK4治疗组、Lenti-NK4治疗组、hBMSCs-GFP对照组和PBS对照组,分别在分组后0、7、14、21天尾静脉注射给药;测量瘤体大小,按πabc/6计算体积;HE染色观察组织病理学变化,免疫组织化学方法(Immunohis tochemistry,IHC)检测CD31、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)表达,原位缺口末端标记法(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling,TUNEL)检测细胞凋亡。结果:
     1、分离培养的hBMSCs流式细胞术检测显示CD44、CD105阳性,而CD34、CD45阴性,符合MSCs特征;hBMSCs经骨诱导后茜素红染色阳性。
     2、构建的NK4-EGFP融合基因的重组慢病毒表达载体转入293T细胞后可正常表达;重组慢病毒(Lenti-NK4)滴度为2×108TU/ml;Lenti-NK4转染hBMSCs后荧光表达随着时间延长及MOI值增加而增强、增多;携带NK4基因的hBMSCs(hBMSCs-NK4)NK4表达正常,NK4分泌量随着MOI值增加而增加,同时也随感染时间延长而增加,最佳MOI值为50;MOI为50时,Lenti-NK4转染hBMSCs的转染率达87.8%,Lenti-GFP转染hBMSCs的转染率为96.5%.
     3、4×106MKN45细胞悬液接种与瘤组织块接种皮下移植瘤成瘤率100%,后者成瘤时间明显短于前者(P<0.05);107个瘤组织匀浆细胞悬液腹腔注射,成瘤率100%。
     4.hBMSCs对胃癌趋向性:(1)上室培养hBMSCs的各组中,MKN45、GES-1、空白对照组细胞数分别为239.5±54.3、43.57±4.6、37.3±4.7;MKN45组细胞数明显高于GES-1组及空白对照组(P<0.01),GES-1组与空白对照组无明显差异(P>0.05);上室培养hFB的各组中,MKN45、GES-1、空白对照组细胞数分别为27.7±16.7、16.4±5.1、19.1±6.2,三组相比均无显著差异(P>0.05);下室均培养MKN45时,上室为hBMSCs组迁移至膜对侧细胞数明显高于上室为hFB组(P<0.01),下室均培养GES-1时,上室为hBMSCs组迁移至膜对侧细胞数明显高于上室为hFB组(P<0.05),下室均为空白培养基时,上室为hBMSCs组迁移至膜对侧细胞数明显高于上室为hFB组(P<0.05)。(2)体内实验结果显示注射hBMSCs-GFP的5只裸鼠,瘤体内均可见散在绿色荧光,而注射hFB-GFP的裸鼠,瘤体内未见绿色荧光,注射Lenti-GFP的5只裸鼠,仅有1例瘤体内可见散在荧光。hBMSCs-GFP组瘤体内荧光表达率明显高于与hFB-GFP及Lenti-GFP组(P<0.01)。hBMSCs-GFP组1例肝脏(20%)及1例肺(20%)有散在荧光,与瘤体内荧光表达率相比明显低(P<0.05),心、肾、脾中未见GFP表达(P<0.01)。
     5、以hBMSCs为载体的NK4基因治疗对胃癌治疗效果:(1)hBMSCs-NK4对皮下移植瘤治疗结果显示hBMSCs-NK4治疗组瘤体体积均明显小于hBMSCs-GFP及PBS组(P<0.05);Lenti-NK4治疗组瘤体体积在各时段与PBS组相比无显著性差异(P>0.05),在第3周时小于hBMSCs-GFP组(P<0.05),其余时间与hBMSCs-GFP组相比无显著性差异(P>0.05);hBMSCs-NK4治疗组抑瘤率达52.2%,Lenti-NK4治疗组抑瘤率为29.4%。(2)hBMSCs-NK4组瘤重1.94±0.67g,明显低于hBMSCs-GFP组及PBS组(P<0.05):Lenti-NK4组瘤重2.16±0.49g,低于hBMSCs-GFP组及PBS组瘤重,但无显著性差异(P>0.05);(3)hBMSCs-NK4组坏死评分3.63±0.47,明显低于两对照组(P<0.05);Lenti-NK4组肿瘤坏死评分为3.25±0.38,与两对照组相比无明显差异(P>0.05):(4)hBMSCs-NK4组微血管密度(microvessel density,MVD)为5.6±1.47,明显低于hBMSCs-GFP和PBS组(P<0.05),;Lenti-NK4组瘤组织MVD低于hBMSCs-GFP和PBS组,但三组相比均无显著性差异(P>0.05):(5)hBMSCs-NK4和Lenti-NK4组凋亡指数(Apoptotic index,Al)分别为7.31±1.90、4.2±1.3,对照组hBMSCs-GFP和PBS组AI分别为2.69±0.55、1.95±0.91,hBMSCs-NK4组AI均显著高于Lenti-NK4组及两对照组(P<0.01),Lenti-NK4组AI亦显著高于两对照组(P<0.05)。
     结论:
     1、采用全骨髓培养法可分离获得高纯度的hBMSCs;
     2、携带NK4的慢病毒能安全、有效地转染hBMSCs,转染后NK4基因可持续稳定表达;
     3、hBMSCs在体内外对胃癌均有明显趋向性;
     4、hBMSCs为载体的NK4基因治疗抑制肿瘤血管形成及诱导肿瘤细胞凋亡使肿瘤组织坏死增加,从而抑制胃癌皮下移植瘤的生长。MSCs可能成为胃癌靶向基因治疗的有效细胞载体。
Objective:
     1. To establish the experimental method for isolating, culturing, and proliferating human bone-derived mesenchymal stem cells (hBMSCs) in vitro;
     2. To construct NK4 and enhance green fluorescent protein fuse gene of recombinant lentiviral expression vector;
     3. To investigate the tropism of hBMSCs to gastric cancer in vitro and in vivo;
     4. To observation the treatment effectiveness of lentiviral vector mediated NK4 gene-modified bone-derived mesenchymal stem cell for gastric cancer and its related mechanism.
     Methods:
     1. hBMSCs were isolated and expanded in culture by bone marrow direct plating method and identified by flow cytometry detecting CD34、CD44、CD45、CD 105 and the ability to differentiate into osteocytes; Calcium deposition was detected using alizarin red stain;
     2. NK4 gene was cloned from HGFcDNA by polymerase chain reaction and identified by gene sequencing. NK4 and enhance green fluorescent protein fuse gene of recombinant lentiviral expression vector was constructed by In-Fusion technique and the expression of GFP was detected by Western blot and observated by fluorescence microscope.The lentiviral titer was detected by real-time quantitative PCR(RT Q-PCR).
     3. After lentiviral vector expressing NK4 and EGFP fuse gene (Lenti-NK4) and lentiviral vector expressing EGFP (Lenti-GFP) transfected hBMSCs, the optimum(Multiplicity of infection,MOI) was determined by the expression of fluorescence and the transfection efficiency. The expression of NK4 was determined by enzyme linked immunosorbent assay (ELISA) and Western blot.
     4. To set up tumor xenografts model, MKN45 tumor cells suspension and tumor tissues were inoculated subcutaneously into athymic nude mice; to set up tumor implantation metastasis model, MKN45 tumor cells suspension and tumor tissues cells suspension were injected into the peritoneal cavity of athymic nude mice.
     5. (1) Cell migration assay was done to determine the tropism of hBMSCs to gastric cancer cells in vitro by Transwells co-culture system. MKN45, GES-1 or culture media were plated on the bottom well of a Transwell plate for 24 h before hBMSCs or hFB were added to the top well. Then the top wells were removed after 24 h and the migrated cells on the bottom side were fixed, stained using crystal violet, and counted in microscope. (2)After subcutaneous tumors had become established, the athymic nude mice were separated into three groups randomly and hBMSCs-GFP, hFB-GFP and Lenti-GFP were injected into tail vein. The athymic nude mice were sacrificed after seven days, the expression of GFP in the tumors and each organ was observed in fluorescence microscope.
     6. After subcutaneous tumors had become established, thirty two athymic nude mice were separated into four groups randomly, then hBMSCs-NK4, Lenti-NK4, hBMSCs-GFP and PBS were injected into tail vein at 0,7,14. and 21 d. The tumors were measured and volume of tumor was calcutated as n abc/6. The histopathology of tumors was observed by hematoxylin and eosin stain; CD31 and PCNA were detected by immunohistochemistry and apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.
     RESULTS
     1. The signs of hBMSCs with positive CD44, CD 105 and negative CD34, CD45 were detected by the flow cytometry, and that is consisted with the characteristics of MSCs. hBMSCs were cultured in medium supplemented with components for osteogenic differentiation. Cells were Alizarin red stain 21 d later. Red-stained calcium deposits were detected in differentiated cultured hBMSCs.
     2. Recombinant lentiviral expression vector of NK4 and enhance green fluorescent protein fuse gene could express correctly after transfected 293T cells. The titer of Lenti-NK4 is 2x108TU/ml. With time extension and MOI increased, green fluorescence and the secretory volume of NK4 in culture supernatant increased gradually after Lenti-NK4 transfected hBMSCs. The optimum MOI of hBMSCs was 50.The transfection efficiency was 87.8% or 96.5% when Lenti-NK4 or Lenti-GFP transfected hBMSCs with MOI 50.
     3. All the mice implanted subcutaneously with 4×106/ml MKN45 cell suspensions or tumor tissues developed subcutaneous tumor nodules and time of tumor formation in the latter method was shorter than in the former method significantly (P<0.05). All the mice injected with 107/ml cells suspension of tissue homogenate into the peritoneal cavity developed metastasis foci.
     4. (1) Directional migration of hBMSCs through membranes were significantly stimulated by human gastric cancer cells MKN45 in vitro(239.5±54.3 migrated cells compared with GES-1 and medium 43.57±4.6,37.3±4.7) (P<0.01). There was not significantly different between GES-1 and medium (P>0.05). Directional migration of hFB through membranes were not significantly stimulated by human gastric cancer cells MKN45 in vitro(27.7±16.7, migrated cells compared with GES-1 and blank control 16.4±5.1,19.1±6.2) (P>0.05). hBMSCs migrating to MKN45,GES-1 and medium increased significantly compared with hFB (P<0.05). (2) Fluorescent hBMSCs were found within the tumor tissue. No fluorescent signal in tumors was visible in cryosections from hFB control group. Fluorescence in tumor tissue was observed in 1 of 5 mice in Lenti-GFP group. There were significantly differences among three groups (P<0.01). Fluorescence in liver and lung was observed in 1 of 5 mice injected hBMSCs-GFP. There was no fluorescence in heart, kidney and spleen in all mice injected hBMSCs-GFP and the positive rate of fluorescence in tumor was significantly higher than each organ (P<0.05)
     5、(1) The volume of tumor in hBMSCs-NK4 group was significant small compared with hBMSCs-GFP and PBS groups (P<0.05). The volume of tumor in Lenti-NK4 group was not significant small compared with PBS group (P>0.05) and hBMSCs-GFP group in the first, second and fourth week (P>0.05).The inhibition ratio of tumor was 52.2% and 29.4% in hBMSCs-NK4 and Lenti-NK4 groups. (2) The weigh of tumor in hBMSCs-NK4 was 1.94±0.67g significantly lower than hBMSCs-GFP and PBS groups (P<0.05). There were no significant differences among Lenti-NK4, hBMSCs-GFP and PBS groups (P>0.05). (3) The score of tumor necrosis was 3.63±0.47 in hBMSCs-NK4 group significantly lower than hBMSCs-GFP and PBS groups (P<0.05). There were no significant differences among Lenti-NK4, hBMSCs-GFP and PBS groups (P>0.05). (4) The microvessel density of tumor was 5.6±1.47 in hBMSCs-NK4 group significantly lower than hBMSCs-GFP and PBS groups (P<0.05). There were no significant differences among Lenti-NK4, hBMSCs-GFP and PBS groups (P>0.05).(5) Apoptotic index of tumor cells was 2.69±0.55,1.95±0.91 in hBMSCs-GFP and PBS groups, whereas it significantly increased to 7.31±1.90 in hBMSCs-NK4 groups (P<0.01) and 4.2±1.3 in Lenti-NK4 groups (P<0.05)
     CONCLUSIONS:
     1. Highly purified hBMSCs can be obtained by by bone marrow direct plating method.
     2. hBMSCs can be transfected by lentiviral vector expressing NK4 gene safely and efficiently and the expressin and secretion of NK4 is stable.
     3. The tropism of hBMSCs to gastric cancer is obvious in vitro and in vivo.
     4. NK4 gene therapy using hBMSCs as vehicle can inhibit angiopoiesis of tumors and induce tumor cells apoptosis and make tumor necrosis increase, then inhibit gastric cancer growth. MSCs may be suitable delivery vehicles for gastric cancer targeted therapy.
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