Enhanced therapeutic effects of combined chemotherapeutic drugs and midkine antisense oligonucleotides for hepatocellular carcinoma

AIM： To evaluate the effect of combined antisense oligonucleotides targeting midkine （MK-AS） and chemotherapeutic drugs [cisplatin（DDP）, 5-fluorouracil （5-FU） and adriamycin （ADM）] on inhibition of HepG2 cell proliferation, and to analyze the efficacy of MK-AS used in combined ADM in in situ human hepatocellular carcinoma （HCC） model. METHODS： HepG2 cells were treated with MK-AS and/or chemotherapeutic drugs mediated by Lipofectin, and cell growth activity was determined by MTS assay. An in situ HCC model was used in this experiment. MK- AS, ADM and MK-AS ＋ ADM were given intravenously for 20 d, respectively. The animal body weight and their tumor weight were measured to assess the effect of the combined therapy in vivo. RESULTS： Combined treatment with MK-AS reduced the IC50 of DDP, 5-FU and ADM in HepG2 cells. MK-AS significantly increased the inhibition rate of DDP, 5-FU and ADM. Additionally, synergism （Q 1.15） occurred at a lower concentration of ADM, 5-FU and DDP with combined MK-AS. Combined treatment with MK-AS and ADM resulted in the more growth inhibition on in situ human HCC model compared with treatment with chemotherapeutic drugs alone. CONCLUSION： MK-AS increases the chemosensitivity in HepG2 cells and in situ human HCC model, and the combination of MK-AS and ADM has a much better in vitro and in vivo synergism.     

MK-siRNA对人乳腺癌细胞的生长、粘附及迁移能力的影响

目的:探讨siRNA MK转染对乳腺癌MCF-7细胞增殖和转移能力的影响。方法:构建特异性抑制Midkine基因siRNA片段,转染至乳腺癌MCF-7细胞中,CCK-8法比较细胞增殖能力的改变,体外迁移实验比较细胞运动能力的改变,基质胶粘附实验比较细胞粘附率的变化。结果:MK表达下调后,MCF-7细胞的增殖能力明显降低(P<0.05),体外迁移能力显著降低(P<0.05),siRNA细胞对基质胶的粘附率明显降低(P<0.05)。结论:MK基因表达下调能降低乳腺癌细胞迁移运动和降低细胞对基质胶的粘附率,并降低肿瘤细胞的增殖,提示MK在乳腺癌恶性进展中起重要作用。     

Midkine mRNA Is Overexpressed in Pancreatic Cancer

Purpose Midkine (MK) has been reported to be a possible molecular marker for the diagnosis of pancreatic cancer. We investigated the feasibility of quantitative analysis of MK mRNA by quantitative real-time RT-PCR (qRT-PCR) as a promising tool for the diagnosis of pancreatic cancer. Results We found that pancreatic cancer tissues expressed significantly higher levels of MK mRNA than intraductal pancreatic mucinous neoplasm (IPMN) and non-neoplastic pancreatic tissues (P < 0.05); in contrast, we did not find any differences in MK mRNA expression between IPMN and non-neoplastic pancreatic tissues. Additionally, we observed that poorly differentiated carcinoma samples expressed higher levels of MK mRNA than well-differentiated carcinoma samples, although a significant difference was not observed. Conclusions The present data suggests that quantitative analysis of MK mRNA provides an objective and sensitive evaluation and may be a promising modality for the diagnosis of pancreatic cancer and the prediction of its prognosis.     

人Midkine启动子驱动的HSV-TK自杀基因体外抗肝癌作用

目的 观察以人Midkine(MK)启动子调控单纯疱疹病毒胸苷激酶基因(HSV-TK)/丙氧鸟苷(GCV)自杀基因系统在体外对人肝癌细胞的杀伤效应.方法 以含有人MK启动子调控的HSV-TK基因的重组腺病毒分别感染体外培养的甲胎蛋白(AFP)阳性人肝癌细胞BEL-7402和AFP阴性人肝癌细胞SMMC-7721,RT-PCR法检测HSV-TK基因在上述两种细胞中的转录表达,观察GCV对人肝癌细胞的杀伤作用.结果 GCV在体外对重组腺病毒感染的BEL-7402及SMMC-7721均有明显的杀伤作用,又以前者为著.相同的病毒滴度,其杀伤作用随着GCV浓度的增高而增强.均具有旁观者效应.结论 在体外表现HSV-TK基因的AFP阳性及阴性人肝癌细胞均可被人MK启动子调控的自杀基因HSV-TK杀伤.     

Increased Midkine Gene Expression in Human Gastrointestinal Cancers

Midkine (MK) is a product of a retinoic acid-responsive gene, and is a novel growth differentiation factor. We examined the expression of the MK gene in specimens of 47 surgically removed human carcinomas of the gastrointestinal organs, namely, gastric, colorectal, hepatocellular, pancreatic, esophageal, ampullary duodenal and bile duct carcinomas. In most cases, the MK mRNA level was higher in cancer specimens than in the corresponding non-cancerous tissues. Furthermore, MK mRNA was more highly expressed in the colon adenocarcinoma lesion than in the adenoma lesions, in the two familial polyposis cases. While MK mRNA was not detected in the normal liver, it became detectable in cirrhotic tissues in 2 of 4 cases, and its expression was increased in the cancerous tissues. Thus, the increase of MK mRNA level is a phenomenon seen in many human gastrointestinal carcinomas. The increased expression of the MK gene in gastric carcinoma was significantly more prominent in well and moderately differentiated adenocarcinomas than in poorly differentiated adenocarcinomas and signet ring cell carcinomas.     

MK、CD105及VEGF在大肠癌组织中的表达及其临床意义

目的探讨MK、CD105和VEGF的表达与大肠癌生物学行为的关系及对预后的意义。方法采用免疫组织化学方法检测50例大肠癌组织MK、CD105和VEGF表达水平,并对其与大肠癌临床病理特征的关系进行统计学分析。结果本组50例大肠癌组织中,MK阳性表达率为72%,VEGF的阳性表达率为64%,MK和VEGF均为阳性时CD105标记的大肠癌组织MVD值(73.87±6.13)明显高于MK和VEGF均为阴性组的MVD值(62.94±6.99)(P<0.01)。三者的表达均与大肠癌的Dukes分期、淋巴结转移、浸润深度有关。结论MK的阳性表达与大肠癌的发生、发展和预后密切相关,可联合VEGF和CD105作为1组有价值的肿瘤标记和预后指标。     

Overexpression of Midkine in Pancreatic Duct Adenocarcinomas Induced by N-Nitrosobis(2-oxopropyl)amine in Hamsters and Their Cell Lines

The expression of midkine (MK) was investigated in pancreatic ductal hyperplasias, atypical hyperplasias and adenocarcinomas induced by N -nitrosobis(2-oxopropyl)amine (BOP) in hamsters, and in hamster ductal adenocarcinoma cell lines (HPD-1NR, -2NR and -3NR). MK mRNA was clearly overexpressed in invasive pancreatic duct adenocarcinomas (PCs) and the three cell lines as assessed by northern blot analysis, and MK protein expression increased from ductal hyperplasia through atypical hyperplasias, intraductal carcinomas and invasive PCs by immunohistochemistry. The extent of overexpression of MK mRNA in PCs was almost the same as in hamster whole embryonic tissue. MK is reported to be a retinoid-responsive gene, but MK mRNA expression was not affected by treatment with all- trans retinoic acid (tRA) or N -(4-hydroxyphenyl)retinamide (4- HPR) in HPD-1NR cells. The results thus suggest that MK expression is involved in the development and progression of pancreatic ductal adenocarcinomas induced by BOP in hamsters, with loss of upregulation by retinoic acid.     

胰腺癌组织中Midkine的表达与细胞增殖及凋亡的关系

目的研究Midkine(MK)蛋白在人胰腺癌组织中的表达与肿瘤细胞增殖和凋亡之间的关系及意义。方法收集人胰腺癌组织标本49例,免疫组化SP法检测MK蛋白和增殖细胞核抗原Ki67的表达,原位末端标记(TUNEL)法检测细胞凋亡指数,另取同期15例正常胰腺组织作对照。结果15例正常胰腺组织中均未发现MK和Ki67阳性表达,细胞凋亡较少(0.63±0.21);77.1%(35/49)的胰腺癌组织中有MK阳性表达,其表达与肿瘤组织学分级、临床分期和淋巴结转移有关(P<0.05);49例胰腺癌组织中Ki67平均标记指数(LI)为(31.67±15.41),平均凋亡指数(AI)为(5.93±4.18);MK表达阳性组Ki67平均标记指数为(35.27±16.03),显著高于阴性组的(22.67±13.66)(P<0.05);MK表达阳性组凋亡指数为(5.13±2.69),显著低于阴性组的(7.93±6.65)(P<0.05)。结论MK蛋白可作为反映胰腺癌生物学行为的指标之一,其在胰腺癌中的表达可能起到促进肿瘤细胞增殖和抑制凋亡的作用,MK基因可能成为胰腺癌治疗的新靶点。     

肝细胞癌高表达中期因子蛋白与肝内转移的关系

目的 探讨中期因子（midkine,MK)蛋白在肝细胞癌（hepatocellular carcinoma,HCC）中的表达特点、水平及其与肝内侵袭、转移的可能关系。方法 应用免疫组织化学染色、Western印迹等方法,对33例人HCC组织、10例良性肝肿瘤组织及其配对瘤旁肝组织进行了MK蛋白定位表达及表达水平的研究,并结合肝内有无卫星灶形成进行分析。结果 免疫组织化学结果与Western印迹结果具有一致性（P＞0．05）。在正常肝组织及良性肝病变组织中,MK无表达;而在HCC组织中,MK高表达。在癌旁肝硬化组织中,MK微量表达;而在肿瘤侵袭前沿区域,MK表达则增强。HCC中MK表达率与其组织学类型、组织分级、瘤体大小、包膜状况、AFP值之间差异均无显著性（P＞0．05）,但在有卫星灶形成的HCC中,MK蛋白表达率显著高于无卫星灶形成者（P＜0．05）。结论 HCC在蛋白水平上表达MK增加,并可能与肝内侵袭、转移有关。     

中期因子对骨折愈合过程微血管生成影响的实验研究

目的 探讨中期因子对骨折端微血管生成对骨折愈合的影响。 方法 采用兔尺骨骨折模型,给以中期因子持续刺激骨折端7天为实验组,给予生理盐水刺激的为对照组,然后在21、42、63、84天,分别获取标本。采用组织学Lane-sandhu评分分析骨组织的生成;免疫组化分析骨折端VEGF和CD34表达来探讨骨折端的微血管生长情况,采用图像分析和MVD评分进行统计分析。 结果 组织学Lane-sandhu评分在42和63天实验组明显高于对照组,VEGF表达在42和63天实验组明显高于对照组;而CD34在42、63、84天周实验组明显高于对照组。 结论 中期因子通过刺激骨折端微血管增生增加是骨折端骨痂生长加快的原因,有利于骨折愈合。