沉默chk1基因对人食管癌细胞化疗增敏作用的研究
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摘要
我国食管癌的发病率居世界首位,而河南省太行山区又是我国食管癌的高发地区。其具有侵袭性强、进展迅速、易复发且死亡率高等特点。目前,食管癌的治疗方法主要包括手术切除、化疗、放疗以及生物治疗等,其中化疗是治疗中晚期食管癌患者的主要手段之一。但在临床治疗过程中许多患者出现化疗抵抗,其原因主要是因为常规化疗药物可以激活肿瘤细胞周期检测点,从而通过相关信号传导通路,使受损DNA的自我修复,从而逃避凋亡。
细胞周期检测点的激活是一种细胞的自我保护机制。当细胞受到化疗等因素损伤后,可激活多个DNA损伤周期检测点(G1/S、S、G2/M),将受损的细胞停滞于相应的检测点DNA修复,从而避免过多的细胞产生凋亡。在细胞周期检测点信号传导通路中细胞周期检测点激酶1(checkpoint kinase1, Chk1)起着重要作用。Chk1是细胞周期检测点最重要的丝氨酸/苏氨酸激酶,主要参与G2/M期细胞周期检测点信号传导。
Chk1可能是迄今最合适消除G2期阻滞的靶分子。目前国内外学者主要采用Chkl siRNA及反义寡核苷酸技术沉默肿瘤细胞中Chk1表达,达到增强恶性肿瘤细胞放疗敏感性的目的。有关通过沉默Chk1达到增强恶性肿瘤细胞化疗敏感性的报道甚少,通过沉默Chk1达到增强食管癌细胞化疗敏感性的研究尚未见报道。
顺铂是目前恶性肿瘤化疗的基本药物之一。然而大量的临床研究发现,含铂类药物的联合化疗方案的临床有效率明显降低,主要原因是由铂类引发的多药耐药即化疗抵抗,这也成为制约晚期恶性肿瘤临床治疗得主要原因。目前大量研究发现,常规化疗药物可造成肿瘤细胞不同类型的DNA损伤,进而激活DNA损伤检测点,促进肿瘤细胞DNA损伤修复,从而使细胞逃避凋亡。因此,DNA损伤检测点的功能状态在一定程度上决定了化疗的疗效好坏。本研究拟通过Chk1siRNA沉默食管癌细胞中Chk1的表达,体内外观察Chk1基因沉默后顺铂对食管癌化疗敏感性的影响,为临床增强食管癌等恶性肿瘤化疗敏感性奠定理论基础。本研究共分以下4个部分。
第一部分Chk1在食管鳞癌组织中的表达及其意义
方法
1.采用免疫组织化学、Western blot、原位杂交、RT-PCR等技术分别检测食管鳞癌组织(62例)、癌旁不典型增生组织(31例)及正常食管黏膜组织(62例)中Chk1蛋白及mRNA的表达情况。
2.统计学方法：应用SPSS13.0软件进行数据处理。采用x2检验、t检验和方差分析,并利用Spearman进行相关性分析。检验水准a=0.05。
结果
1.Chk1蛋白阳性表达主要定位于食管癌细胞的胞核及胞质内,呈棕黄色颗粒；Chk1mRNA阳性表达定位于食管癌细胞的胞质内,呈蓝紫色颗粒；二者在癌旁不典型增生组织及正常食管黏膜组织内阴性表达或低表达。
2.免疫组化、Western blot及原位杂交、RT-PCR联合检测结果显示：在正常食管黏膜、癌旁不典型增生和食管鳞癌组织中,Chk1蛋白的阳性表达率依次升高,三者间两两相比差异均有统计学意义(P<0.05)；其mRNA表达水平亦依次升高,三者组间比较差异有统计学意义(P＜0.05)。
3.在深层浸润组食管鳞癌组织中,Chk1蛋白、mRNA的表达均显著高于浅层浸润组,两组相比差异有统计学意义(P<0.05)。
4.在有淋巴结转移组的食管鳞癌组织中,Chk1蛋白、mRNA的表达显著高于无淋巴结转移组,两组相比差异有统计学意义(P<0.05)。
5.随着食管鳞癌分化级别的升高,鳞癌组织中Chk1蛋白、mRNA的表达依次升高,组间两两相比差异有统计学意义(P＜0.05)
6.Chk1蛋白mRNA的表达与食管鳞癌患者的性别、年龄无关(P＞0.05)。
第二部分Chk1siRNA表达载体的构建
方法
1.构建pSilencer3.1-Chk1siRNA重组载体。
2.将3个已构建好的Chk1siRNA载体和一个阴性对照siRNA载体分别转染食管鳞癌EC9706细胞,并获得稳定细胞株。
3.应用实时荧光定量PCR检测细胞中Chkl mRNA的相对表达量；运用Western blot方法检测细胞中Chk1蛋白的相对表达,挑选抑制效果最好的Chk1siRNA。
4.统计学处理：应用SPSS11.0软件处理,采用x2检验、t检验和方差分析。检验水准α=0.05。
结果
1.成功构建了重组载体pSilencer3.1-Chkl siRNA1、pSilencer3.1-Chk1siRNA2和pSilencer3.1-Chkl siRNA3。
2.三个Chk1siRNA载体转染食管癌EC9706细胞后,Chk1蛋白、mRNA的表达水平均显著低于未处理的EC9706细胞和对照siRNA组(P<0.05)。
3.三个Chk1siRNA组之间相比,Chk1siRNA1和Chk1siRNA3之间Chk1
mRNA表达无差异(P＞0.05),但均显著高于Chk1siRNA2组中Chk1mRNA的表达水平(P＜0.05)。
第三部分Chk1siRNA干扰表达载体体外对顺铂抑制人食管癌EC9706细胞增殖、诱导其凋亡的影响
方法
1.食管癌EC9706细胞分组培养,即A组：正常组,未经任何处理：B组：Chk1siRNA2载体转染人食管鳞癌EC9706细胞24h后,再应用浓度为1Oμmol／L的顺铂再处理EC9706细胞12h；C组：10μmo1／L顺铂处理EC9706细胞12h。
2.采用免疫细胞化学、Western blot和原位杂交、RT-PCR方法检测各组细胞中Chk1蛋白和mRNA的表达。
3.采用MTT方法观察各组细胞增殖情况。
4.运用TUNEL法、流式细胞术等方法检测各组细胞凋亡情况。
5.统计学处理：应用SPSS13.0软件处理,计数资料采用x2检验、计量资料行t检验或方差分析,检验水准a=0.05。
结果
1.MTT法检测结果：B组(Chk1siRNA2+顺铂)与C组(顺铂)及A组(正常对照组)相比,细胞生长抑制率明显增加,差异均有统计学意义(P<0.05)。
2.免疫细胞化学及Western blot检测结果：B组(Chk1siRNA2+顺铂)与C组(顺铂)及A组(正常对照组)相比,食管癌EC9706细胞中Chk1蛋白表达明显下调,组间两两相比差异均有统计学意义(P＜0.05)。
3.原位杂交及RT-PCR检测结果：B组(Chk1siRNA2+顺铂)细胞中Chk1mRNA表达明显低于C组(顺铂)及A组(正常对照组),组间两两相比差异均有统计学意义(P＜0.05)。
4. TUNEL方法检测凋亡结果：B组(Chk1siRNA2+(?)顺铂)细胞中凋亡指数(AI)明显高于C组(顺铂)及A组(正常对照组),组间两两相比差异均有统计学意义(P＜0.05)。
5.流式细胞仪检测凋亡结果：B组(Chk1siRNA2+顺铂)早期凋亡细胞数
和晚期凋亡细胞数均较C组(顺铂)及A组(正常对照组)明显增加,且差异均有统计学意义(P均＜0.05)。
第四部分Chk1siRNA干扰表达载体体内对顺铂诱导的食管癌移植瘤细胞生物学行为的影响
方法
1.培养食管癌EC9706细胞,构建裸鼠食管癌移植瘤模型。
2.分组饲养荷瘤裸鼠,即A组：正常组,瘤体注射无菌生理盐水；B组：瘤体注射Chk1siRNA2加浓度为(5mg/kg)的顺铂；C组：瘤体注射顺铂(5mg／kg)。各组均每3天1次,共5次。
3.观察各组移植瘤的生长状况。
4.采用免疫组化、Western blot和原位杂交、RT-PCR方法检测各组移植瘤组织中Chk1蛋白和mRNA的表达。
5.运用TUNEL法方法检测各组细胞凋亡情况。
6.统计学处理：应用SPSS13.0软件处理,计数资料采用x2检验、计量资料行t检验或方差分析,检验水准a=0.05。
结果
1.B组(Chk1siRNA2+J顷铂)移植瘤体积明显小于C组(顺铂)及A组(正常对照组),组间两两相比,均有统计学差异(P＜0.001)。
2.免疫组化及Western blot检测结果：B组(Chkl siRNA2+顺铂)食管癌移植瘤组织中Chk1蛋白表达明显低于C组(顺铂)及A组(正常对照组),组间两两相比差异均有统计学意义(P＜0.05)。
3.原位杂交及RT-PCR检测结果：B组(Chkl siRNA2+(?)顺铂)移植瘤组织中Chk1mRNA表达显著低于C组(顺铂)及A组(正常对照组),组间两两相比差异均有统计学意义(P＜0.05)。
4. TUNEL方法检测结果：B组(Chk1siRNA2+(?)顺铂)细胞中凋亡指数(AI)明显高于C组(顺铂)及A组(正常对照组),组间两两相比差异均有统计学意义(P＜0.05)。
全文结论
1.Chk1在食管鳞癌组织中存在高表达。
2.Chk1蛋白及mRNA表达与食管鳞癌发生发展及浸润转移有关。
3.成功构建了三个Chkl siRNA载体。
4. Chkl siRNA2具有最好的抑制效果。
5. Chkl siRNA干扰表达载体可体外下调人食管癌EC9706细胞中Chkl蛋白及mRNA表达。
6.沉默Chk1表达可体外增强顺铂抑制人食管癌EC9706细胞增殖、诱导其凋亡的敏感性。
7.成功构建了裸鼠食管癌移植瘤模型。
8.沉默Chk1表达,可体内增强顺铂对裸鼠食管癌移植瘤生长抑制,诱导移植瘤细胞凋亡的效应。
全文总结论
1.Chk1在食管鳞癌组织中存在高表达。
2.Chk1蛋白及mRNA表达与食管鳞癌发生发展及浸润转移有关。
3.成功构建了三个Chkl siRNA载体。
4. Chkl siRNA2具有最好的抑制效果。
5. Chkl siRNA2可体外下调人食管癌EC9706细胞中Chk1蛋白及mRNA表达。
6.沉默Chk1表达可体外增强顺铂抑制人食管癌EC9706细胞增殖、诱导其凋亡的敏感性。
7.成功构建了裸鼠食管癌移植瘤模型。
8.沉默Chk1表达,可体内增强顺铂对裸鼠食管癌移植瘤的生长抑制,促进其诱导移植瘤细胞凋亡的效应。
The incidence of esophageal carcinoma in our country ranks first in the world, and the Taihang Mountain area in Henan province is high risk area for esophageal carcinoma in China. Its attack is strong, rapid, easy to relapse and mortality high. Currently, the treatment of esophageal carcinoma includes surgery, chemotherapy, radiation therapy, biological therapy and so on, in which chemotherapy is one of the principal means of treatment in patients with advanced esophageal carcinoma. But in the clinical course of treatment, many patients resistant to chemotherapy. Study found that chemotherapy drugs can activate cell cycle checkpoint signaling pathway, causing tumor cells to self-healing of damaged DNA to apoptosis escape and cause chemotherapy resistance.
Activation of cell cycle checkpoint is a cellular self-protection mechanism. After the damage of chemotherapy and other factors, cells can activate multiple DNA damage cycle checkpoints (G1/S, S and G2/M) of the destroyed cells stagnation in the detection point of DNA repair, thus avoiding excessive chilliness apoptosis. Cell cycle checkpoint kinase1(Checkpoint kinase1, Chkl) plays an important role in the cell cycle checkpoint signaling pathway. Chkl is the most prominent point of the cell cycle of the serine/threonine kinase, mainly involved in signal transduction during the G2/M cell cycle.
Chkl may be the most suitable target molecules to eliminate G2arrest by far. Contemporary scholars at home and abroad mainly used Chkl siRNA and antigens oligonucleotide technology to silence the expression of Chkl in tumor cells to enhance the radiosensitivity of malignant tumor cells. Reports about silencing Chkl to enhance the sensitivity of tumor cell to chemotherapy are little, and the study which through silencing Chkl to enhance the sensitivity of esophageal carcinoma cell has not been reported.
Cisplatin is one of the essential drugs for treatment of esophageal malignancy. However, multiple resistance, caused by platinum-based, makes the clinical efficiency of joint programs which with platinum-based decreased significantly, that became a bottleneck in the clinical progress in the treatment of advanced malignant tumor. Nowadays, clinical and in vitro and in vivo experiments has researched to their resistance extensively, recently found that the chemotherapy drugs cause different types of DNA damage in tumor cells, activated the DNA damage checkpoint, leading to tumor cell DNA damage repair, so that the cell avoiding apoptosis. Therefore, the efficacy of chemotherapy is to a large extent depends on the functional status of the DNA damage checkpoint. This study is proposed by Chkl siRNA silencing the expression of Chkl in esophageal carcinoma cells, observation in vitro and in vivo, the effect of cisplatin on the chemotherapy sensitivity of esophageal carcinoma after the Chkl gene silencing and that lay the theoretical foundation for clinical for the enhanced sensitivity to chemotherapy of malignant tumor of esophageal carcinoma. This research is divided into the following4parts.
Part I Chkl expression in esophageal squamous cell carcinoma and its significance
Methods:
1. Using immunohistochemistry, Western blot, in situ hybridization, RT-PCR, to detect the expression of RhoC gene in62cases of esophageal squamous cell carcinoma,31cases of adjacent dysplasia and62cases of normal esophageal mucosa.
2. Statistical analysis:Statistics analysis was utilized by SPSS13.0software, using chi-square test, T test and variance analysis and Spearmanm, test standard α=0.05.
Results:
1. Expression of Chk1protein was predominantly localized in esophageal cancer cell nuclei and cytoplasm, brown yellow granules; Chk1mRNA positive expression was localized in the esophagus cancer cell cytoplasm, purplish blue particles; Chk1protein and Chk1mRNA were an expression no or lower in atypical hyperplasia tissue and normal esophageal mucosa negative.
2. The results of a joint test of immunohistochemistry, Western blot, in situ hybridization, RT-PCR:Chk1mRNA and protein expression level gradually increased from normal esophagus mucous membrane tissue, adjacent atypical hyperplasia tissue to ESCC tissue, and there was significant difference among three groups (P<0.05).
3. Expressions of Chk1mRNA and protein in the deep layer invaded groups were considerably higher than those in infiltrating shallow layer group, and there was significant difference among three groups (P<0.05).
4. Expressions of Chk1mRNA and protein in lymphatic metastasis groups were significantly higher than those without lymphatic metastasis group, and there was significant difference among three groups (P<0.05).
5. Expressions of Chk1mRNA and protein in poor differentiated ESCC tissues were markedly higher than that in well differentiated tissues, and the differences were statistically significant (P<0.05)
6. Expressions of Chk1mRNA and protein were not related to sex and age of the patients with esophagus cancer (P>0.05).
Part Ⅱ Construction of Chk1siRNA expression vector
Methods:
1. Construction of recombinant vector pSilencer3.1-Chk1siRNA.
2. Three pairs of anti-sense oligonucleotide fragments and a pair of non-sense sequence was designed and annealed, and then was introduced into RNA interfering expression vector pSilencer3.1, and the recombinant expression vector pSilencer3.1-Chkl siRNA was successfully constructed.
3. Using RT-PCR to detect the expression of Chkl mRNA in the cells and utilizing of the Western blotting to detect the expression of Chk1protein in the cells, and then pick the best inhibitory effect of Chkl siRNA.
4. Statistical analysis:Statistics analysis was used by SPSS13.0software, using chi-square test, T test and variance analysis and Spearmanm, test standard a=0.05.
Results:
1. Three siRNA expression vector pSilencer3.1-Chkl siRNAl, pSilencer3.1-Chkl siRNA2和pSilencer3.1-Chkl siRNA3were successfully constructed.
2. Three Chkl siRNA vector-transfected EC9706cells, Chkl protein and mRNA expression levels were significantly lower than untreated EC9706cells and control siRNA group (P<0.05).
3. Among the group of Chk1siRNA, compared between Chk1siRNA1and Chkl siRNA3, the expression of Chkl mRNA was no difference (P>0.05), but it was substantially higher than that of Chkl siRNA2Chkl mRNA expression levels (P <0.05).
Part Ⅲ Influence of biology behavior Chkl siRNA interference expression vector for cisplatin induced in vitro of EC9706esophageal cancer cell
Methods:
1. Esophageal cancer cell EC9706group training, namely A groups:normal group, without any treatment; B:Chk1siRNA2carrier transfection people esophageal squamous carcinoma EC9706cell24h, then reprocessing EC9706cell12h using cisplatin of concentration for10μ mol/L; C group:reprocessing EC9706cell12h using cisplatin of concentration for10μ mol/L
2. Using immunohistochemical, Western blot and in situ hybridization, RT-PCR to test the cells Chkl protein and mRNA expression
3. Using MMT method to observe the cell proliferation condition.
4. Using TUNEL and flow cytometry methods to detect cell apoptosis situation.
5.Statistical analysis:Statistics analysis was used by SPSS13.0software, using chi-square test, t test and variance analysis and Spearmanm, test standard α=0.05.
Results:
1. The results of the method of MTT:Compared to the B group (Chkl siRNA2+cisplatin) and C group (cisplatin) and A group (normal control group), the cell growth inhibition rate increased dramatically, the differences were statistically significantly(P <0.05).
2. Immunocytochemistry and Western blot test results:B Group (Chkl siRNA2+cisplatin) and C group (cisplatin) and A group (normal control group), compared with EC9706esophageal cancer cells Chkl protein expression clear cut, two groups of difference in all have statistical significance (P<0.05).
3. The results of In situ hybridization and RT-PCR:the Chkl mRNA expressed in cells in group B(Chkl siRNA2+cisplatin) was significantly lower than the group C(cisplatin) and group A(normal control group), compared two groups of two differences were statistically significant (P<0.05).
4. Apoptosis results of TUNEL method:Cells in group B(Chkl siRNA2+cisplatin) apoptosis index (AI) is obviously higher than that of group C (cisplatin) and A group (normal control group), compared two groups of two differences were statistically significant (P<0.05).
5. Flow cell detection instrument apoptosis results:B Group (Chkl siRNA2+cisplatin) early apoptosis cells number and late apoptosis cells number than the C group (cisplatin) and A group (normal control group) increased considerably, and the differences were statistically significant (P<0.05).
Part Ⅳ The effect of Chk1siRNA interference expression vector on cisplatin induced esophageal carcinoma transplanted tumor cells biological behavior in vivo
Methods:
1. Cultivate esophageal cancer EC9706cell, constructing the nude mouse esophageal cancer transplantation tumor model.
2. Group breeding tumor-burdened nude mouse, namely A groups:normal group, pseudoscience injection Sterile Saline; B group:pseudoscience injection Chk1siRNA2add concentration for (5mg/kg) of cisplatin, C groups:pseudoscience injection cisplatin (5mg/kg). All group every3days one time, a total of5times.
3. Observe the transplantation tumor growth.
4. Using immunohistochemical, Western blot and in situ hybridization, RT polymerase chain reaction (PCR) to test the transplantation of Chkl protein and mRNA expression in the tumor tissues.
5.Using TUNEL method. to test the apoptosis situation of each cell.
6.Statistical analysis:Statistics analysis was used by SPSS13.0software, using chi-square test, T test and variance analysis and Spearmanm, test standard α=0.05.
Results:
1. Transplant pseudoscience product of the B group (Chkl siRNA2+cisplatin) noticeably less than the C group (cisplatin) and A group (normal control group), compared two groups of two, all have statistical difference (P<0.001).
2.The results of immunohistochemical and Western blot test:the expression of Chk1protein in the esophageal cancer transplantation tumor tissues of B group (Chk1siRNA2+cisplatin) was significantly lower than the C group (cisplatin) and A group (normal control group), compared two groups of two, all have statistical difference (P <0.05).
3.The results of in situ hybridization and RT-PCR detection:the expression of Chk1mRNA in the esophageal cancer transplantation tumor tissues of B group (Chk1 siRNA2+cisplatin) was significantly lower than the C group (cisplatin) and A group (normal control group), compared two groups of two, all have statistical difference (P <0.05).
4.The results of TUNEL method detection:the cells apoptosis index (AI) of group B(Chk1siRNA2+cisplatin) is obviously higher than that of the group C (cisplatin) and group A(normal control group), compared two groups of two, all have statistical difference (P<0.05).
Conclusion
1. High levels of Chkl protein and mRNA were observed in esophageal squamous cell carcinoma.
2. The expression of Chkl protein and mRNA were closely associated with the invasion and metastasis of ESCC.
3. Interfering expression vector pSilencer3.1-Chk1siRNA was successfully constructed.
4. Chk1siRNA2has the best inhibiting effect.
5. The expression of Chkl protein and mRNA can be cut by Chkl siRNA interference expression vector in vitro for people with esophageal cancer cells EC9706.
6. Silencing Chkl expression in vitro can enhance the esophageal cancer EC9706cell proliferation and induce its apoptosis sensitivity of which cisplatin on.
7. Successfully constructed nude mouse esophageal cancer transplantation tumor model.
8. Silencing Chkl expression can strengthen the body of nude mouse esophageal cancer transplantation tumor growth inhibition which cisplatin on and induce the effect of cell apoptosis of transplantation tumor.
细胞周期检测点的激活是一种细胞的自我保护机制。当细胞受到化疗等因素损伤后,可激活多个DNA损伤周期检测点(G1/S、S、G2/M),将受损的细胞停滞于相应的检测点DNA修复,从而避免过多的细胞产生凋亡。在细胞周期检测点信号传导通路中细胞周期检测点激酶1(checkpoint kinase1, Chk1)起着重要作用。Chk1是细胞周期检测点最重要的丝氨酸/苏氨酸激酶,主要参与G2/M期细胞周期检测点信号传导。
Chk1可能是迄今最合适消除G2期阻滞的靶分子。目前国内外学者主要采用Chkl siRNA及反义寡核苷酸技术沉默肿瘤细胞中Chk1表达,达到增强恶性肿瘤细胞放疗敏感性的目的。有关通过沉默Chk1达到增强恶性肿瘤细胞化疗敏感性的报道甚少,通过沉默Chk1达到增强食管癌细胞化疗敏感性的研究尚未见报道。
顺铂是目前恶性肿瘤化疗的基本药物之一。然而大量的临床研究发现,含铂类药物的联合化疗方案的临床有效率明显降低,主要原因是由铂类引发的多药耐药即化疗抵抗,这也成为制约晚期恶性肿瘤临床治疗得主要原因。目前大量研究发现,常规化疗药物可造成肿瘤细胞不同类型的DNA损伤,进而激活DNA损伤检测点,促进肿瘤细胞DNA损伤修复,从而使细胞逃避凋亡。因此,DNA损伤检测点的功能状态在一定程度上决定了化疗的疗效好坏。本研究拟通过Chk1siRNA沉默食管癌细胞中Chk1的表达,体内外观察Chk1基因沉默后顺铂对食管癌化疗敏感性的影响,为临床增强食管癌等恶性肿瘤化疗敏感性奠定理论基础。本研究共分以下4个部分。
第一部分Chk1在食管鳞癌组织中的表达及其意义
方法
1.采用免疫组织化学、Western blot、原位杂交、RT-PCR等技术分别检测食管鳞癌组织(62例)、癌旁不典型增生组织(31例)及正常食管黏膜组织(62例)中Chk1蛋白及mRNA的表达情况。
2.统计学方法：应用SPSS13.0软件进行数据处理。采用x2检验、t检验和方差分析,并利用Spearman进行相关性分析。检验水准a=0.05。
结果
1.Chk1蛋白阳性表达主要定位于食管癌细胞的胞核及胞质内,呈棕黄色颗粒；Chk1mRNA阳性表达定位于食管癌细胞的胞质内,呈蓝紫色颗粒；二者在癌旁不典型增生组织及正常食管黏膜组织内阴性表达或低表达。
2.免疫组化、Western blot及原位杂交、RT-PCR联合检测结果显示：在正常食管黏膜、癌旁不典型增生和食管鳞癌组织中,Chk1蛋白的阳性表达率依次升高,三者间两两相比差异均有统计学意义(P<0.05)；其mRNA表达水平亦依次升高,三者组间比较差异有统计学意义(P＜0.05)。
3.在深层浸润组食管鳞癌组织中,Chk1蛋白、mRNA的表达均显著高于浅层浸润组,两组相比差异有统计学意义(P<0.05)。
4.在有淋巴结转移组的食管鳞癌组织中,Chk1蛋白、mRNA的表达显著高于无淋巴结转移组,两组相比差异有统计学意义(P<0.05)。
5.随着食管鳞癌分化级别的升高,鳞癌组织中Chk1蛋白、mRNA的表达依次升高,组间两两相比差异有统计学意义(P＜0.05)
6.Chk1蛋白mRNA的表达与食管鳞癌患者的性别、年龄无关(P＞0.05)。
第二部分Chk1siRNA表达载体的构建
方法
1.构建pSilencer3.1-Chk1siRNA重组载体。
2.将3个已构建好的Chk1siRNA载体和一个阴性对照siRNA载体分别转染食管鳞癌EC9706细胞,并获得稳定细胞株。
3.应用实时荧光定量PCR检测细胞中Chkl mRNA的相对表达量；运用Western blot方法检测细胞中Chk1蛋白的相对表达,挑选抑制效果最好的Chk1siRNA。
4.统计学处理：应用SPSS11.0软件处理,采用x2检验、t检验和方差分析。检验水准α=0.05。
结果
1.成功构建了重组载体pSilencer3.1-Chkl siRNA1、pSilencer3.1-Chk1siRNA2和pSilencer3.1-Chkl siRNA3。
2.三个Chk1siRNA载体转染食管癌EC9706细胞后,Chk1蛋白、mRNA的表达水平均显著低于未处理的EC9706细胞和对照siRNA组(P<0.05)。
3.三个Chk1siRNA组之间相比,Chk1siRNA1和Chk1siRNA3之间Chk1
mRNA表达无差异(P＞0.05),但均显著高于Chk1siRNA2组中Chk1mRNA的表达水平(P＜0.05)。
第三部分Chk1siRNA干扰表达载体体外对顺铂抑制人食管癌EC9706细胞增殖、诱导其凋亡的影响
方法
1.食管癌EC9706细胞分组培养,即A组：正常组,未经任何处理：B组：Chk1siRNA2载体转染人食管鳞癌EC9706细胞24h后,再应用浓度为1Oμmol／L的顺铂再处理EC9706细胞12h；C组：10μmo1／L顺铂处理EC9706细胞12h。
2.采用免疫细胞化学、Western blot和原位杂交、RT-PCR方法检测各组细胞中Chk1蛋白和mRNA的表达。
3.采用MTT方法观察各组细胞增殖情况。
4.运用TUNEL法、流式细胞术等方法检测各组细胞凋亡情况。
5.统计学处理：应用SPSS13.0软件处理,计数资料采用x2检验、计量资料行t检验或方差分析,检验水准a=0.05。
结果
1.MTT法检测结果：B组(Chk1siRNA2+顺铂)与C组(顺铂)及A组(正常对照组)相比,细胞生长抑制率明显增加,差异均有统计学意义(P<0.05)。
2.免疫细胞化学及Western blot检测结果：B组(Chk1siRNA2+顺铂)与C组(顺铂)及A组(正常对照组)相比,食管癌EC9706细胞中Chk1蛋白表达明显下调,组间两两相比差异均有统计学意义(P＜0.05)。
3.原位杂交及RT-PCR检测结果：B组(Chk1siRNA2+顺铂)细胞中Chk1mRNA表达明显低于C组(顺铂)及A组(正常对照组),组间两两相比差异均有统计学意义(P＜0.05)。
4. TUNEL方法检测凋亡结果：B组(Chk1siRNA2+(?)顺铂)细胞中凋亡指数(AI)明显高于C组(顺铂)及A组(正常对照组),组间两两相比差异均有统计学意义(P＜0.05)。
5.流式细胞仪检测凋亡结果：B组(Chk1siRNA2+顺铂)早期凋亡细胞数
和晚期凋亡细胞数均较C组(顺铂)及A组(正常对照组)明显增加,且差异均有统计学意义(P均＜0.05)。
第四部分Chk1siRNA干扰表达载体体内对顺铂诱导的食管癌移植瘤细胞生物学行为的影响
方法
1.培养食管癌EC9706细胞,构建裸鼠食管癌移植瘤模型。
2.分组饲养荷瘤裸鼠,即A组：正常组,瘤体注射无菌生理盐水；B组：瘤体注射Chk1siRNA2加浓度为(5mg/kg)的顺铂；C组：瘤体注射顺铂(5mg／kg)。各组均每3天1次,共5次。
3.观察各组移植瘤的生长状况。
4.采用免疫组化、Western blot和原位杂交、RT-PCR方法检测各组移植瘤组织中Chk1蛋白和mRNA的表达。
5.运用TUNEL法方法检测各组细胞凋亡情况。
6.统计学处理：应用SPSS13.0软件处理,计数资料采用x2检验、计量资料行t检验或方差分析,检验水准a=0.05。
结果
1.B组(Chk1siRNA2+J顷铂)移植瘤体积明显小于C组(顺铂)及A组(正常对照组),组间两两相比,均有统计学差异(P＜0.001)。
2.免疫组化及Western blot检测结果：B组(Chkl siRNA2+顺铂)食管癌移植瘤组织中Chk1蛋白表达明显低于C组(顺铂)及A组(正常对照组),组间两两相比差异均有统计学意义(P＜0.05)。
3.原位杂交及RT-PCR检测结果：B组(Chkl siRNA2+(?)顺铂)移植瘤组织中Chk1mRNA表达显著低于C组(顺铂)及A组(正常对照组),组间两两相比差异均有统计学意义(P＜0.05)。
4. TUNEL方法检测结果：B组(Chk1siRNA2+(?)顺铂)细胞中凋亡指数(AI)明显高于C组(顺铂)及A组(正常对照组),组间两两相比差异均有统计学意义(P＜0.05)。
全文结论
1.Chk1在食管鳞癌组织中存在高表达。
2.Chk1蛋白及mRNA表达与食管鳞癌发生发展及浸润转移有关。
3.成功构建了三个Chkl siRNA载体。
4. Chkl siRNA2具有最好的抑制效果。
5. Chkl siRNA干扰表达载体可体外下调人食管癌EC9706细胞中Chkl蛋白及mRNA表达。
6.沉默Chk1表达可体外增强顺铂抑制人食管癌EC9706细胞增殖、诱导其凋亡的敏感性。
7.成功构建了裸鼠食管癌移植瘤模型。
8.沉默Chk1表达,可体内增强顺铂对裸鼠食管癌移植瘤生长抑制,诱导移植瘤细胞凋亡的效应。
全文总结论
1.Chk1在食管鳞癌组织中存在高表达。
2.Chk1蛋白及mRNA表达与食管鳞癌发生发展及浸润转移有关。
3.成功构建了三个Chkl siRNA载体。
4. Chkl siRNA2具有最好的抑制效果。
5. Chkl siRNA2可体外下调人食管癌EC9706细胞中Chk1蛋白及mRNA表达。
6.沉默Chk1表达可体外增强顺铂抑制人食管癌EC9706细胞增殖、诱导其凋亡的敏感性。
7.成功构建了裸鼠食管癌移植瘤模型。
8.沉默Chk1表达,可体内增强顺铂对裸鼠食管癌移植瘤的生长抑制,促进其诱导移植瘤细胞凋亡的效应。
The incidence of esophageal carcinoma in our country ranks first in the world, and the Taihang Mountain area in Henan province is high risk area for esophageal carcinoma in China. Its attack is strong, rapid, easy to relapse and mortality high. Currently, the treatment of esophageal carcinoma includes surgery, chemotherapy, radiation therapy, biological therapy and so on, in which chemotherapy is one of the principal means of treatment in patients with advanced esophageal carcinoma. But in the clinical course of treatment, many patients resistant to chemotherapy. Study found that chemotherapy drugs can activate cell cycle checkpoint signaling pathway, causing tumor cells to self-healing of damaged DNA to apoptosis escape and cause chemotherapy resistance.
Activation of cell cycle checkpoint is a cellular self-protection mechanism. After the damage of chemotherapy and other factors, cells can activate multiple DNA damage cycle checkpoints (G1/S, S and G2/M) of the destroyed cells stagnation in the detection point of DNA repair, thus avoiding excessive chilliness apoptosis. Cell cycle checkpoint kinase1(Checkpoint kinase1, Chkl) plays an important role in the cell cycle checkpoint signaling pathway. Chkl is the most prominent point of the cell cycle of the serine/threonine kinase, mainly involved in signal transduction during the G2/M cell cycle.
Chkl may be the most suitable target molecules to eliminate G2arrest by far. Contemporary scholars at home and abroad mainly used Chkl siRNA and antigens oligonucleotide technology to silence the expression of Chkl in tumor cells to enhance the radiosensitivity of malignant tumor cells. Reports about silencing Chkl to enhance the sensitivity of tumor cell to chemotherapy are little, and the study which through silencing Chkl to enhance the sensitivity of esophageal carcinoma cell has not been reported.
Cisplatin is one of the essential drugs for treatment of esophageal malignancy. However, multiple resistance, caused by platinum-based, makes the clinical efficiency of joint programs which with platinum-based decreased significantly, that became a bottleneck in the clinical progress in the treatment of advanced malignant tumor. Nowadays, clinical and in vitro and in vivo experiments has researched to their resistance extensively, recently found that the chemotherapy drugs cause different types of DNA damage in tumor cells, activated the DNA damage checkpoint, leading to tumor cell DNA damage repair, so that the cell avoiding apoptosis. Therefore, the efficacy of chemotherapy is to a large extent depends on the functional status of the DNA damage checkpoint. This study is proposed by Chkl siRNA silencing the expression of Chkl in esophageal carcinoma cells, observation in vitro and in vivo, the effect of cisplatin on the chemotherapy sensitivity of esophageal carcinoma after the Chkl gene silencing and that lay the theoretical foundation for clinical for the enhanced sensitivity to chemotherapy of malignant tumor of esophageal carcinoma. This research is divided into the following4parts.
Part I Chkl expression in esophageal squamous cell carcinoma and its significance
Methods:
1. Using immunohistochemistry, Western blot, in situ hybridization, RT-PCR, to detect the expression of RhoC gene in62cases of esophageal squamous cell carcinoma,31cases of adjacent dysplasia and62cases of normal esophageal mucosa.
2. Statistical analysis:Statistics analysis was utilized by SPSS13.0software, using chi-square test, T test and variance analysis and Spearmanm, test standard α=0.05.
Results:
1. Expression of Chk1protein was predominantly localized in esophageal cancer cell nuclei and cytoplasm, brown yellow granules; Chk1mRNA positive expression was localized in the esophagus cancer cell cytoplasm, purplish blue particles; Chk1protein and Chk1mRNA were an expression no or lower in atypical hyperplasia tissue and normal esophageal mucosa negative.
2. The results of a joint test of immunohistochemistry, Western blot, in situ hybridization, RT-PCR:Chk1mRNA and protein expression level gradually increased from normal esophagus mucous membrane tissue, adjacent atypical hyperplasia tissue to ESCC tissue, and there was significant difference among three groups (P<0.05).
3. Expressions of Chk1mRNA and protein in the deep layer invaded groups were considerably higher than those in infiltrating shallow layer group, and there was significant difference among three groups (P<0.05).
4. Expressions of Chk1mRNA and protein in lymphatic metastasis groups were significantly higher than those without lymphatic metastasis group, and there was significant difference among three groups (P<0.05).
5. Expressions of Chk1mRNA and protein in poor differentiated ESCC tissues were markedly higher than that in well differentiated tissues, and the differences were statistically significant (P<0.05)
6. Expressions of Chk1mRNA and protein were not related to sex and age of the patients with esophagus cancer (P>0.05).
Part Ⅱ Construction of Chk1siRNA expression vector
Methods:
1. Construction of recombinant vector pSilencer3.1-Chk1siRNA.
2. Three pairs of anti-sense oligonucleotide fragments and a pair of non-sense sequence was designed and annealed, and then was introduced into RNA interfering expression vector pSilencer3.1, and the recombinant expression vector pSilencer3.1-Chkl siRNA was successfully constructed.
3. Using RT-PCR to detect the expression of Chkl mRNA in the cells and utilizing of the Western blotting to detect the expression of Chk1protein in the cells, and then pick the best inhibitory effect of Chkl siRNA.
4. Statistical analysis:Statistics analysis was used by SPSS13.0software, using chi-square test, T test and variance analysis and Spearmanm, test standard a=0.05.
Results:
1. Three siRNA expression vector pSilencer3.1-Chkl siRNAl, pSilencer3.1-Chkl siRNA2和pSilencer3.1-Chkl siRNA3were successfully constructed.
2. Three Chkl siRNA vector-transfected EC9706cells, Chkl protein and mRNA expression levels were significantly lower than untreated EC9706cells and control siRNA group (P<0.05).
3. Among the group of Chk1siRNA, compared between Chk1siRNA1and Chkl siRNA3, the expression of Chkl mRNA was no difference (P>0.05), but it was substantially higher than that of Chkl siRNA2Chkl mRNA expression levels (P <0.05).
Part Ⅲ Influence of biology behavior Chkl siRNA interference expression vector for cisplatin induced in vitro of EC9706esophageal cancer cell
Methods:
1. Esophageal cancer cell EC9706group training, namely A groups:normal group, without any treatment; B:Chk1siRNA2carrier transfection people esophageal squamous carcinoma EC9706cell24h, then reprocessing EC9706cell12h using cisplatin of concentration for10μ mol/L; C group:reprocessing EC9706cell12h using cisplatin of concentration for10μ mol/L
2. Using immunohistochemical, Western blot and in situ hybridization, RT-PCR to test the cells Chkl protein and mRNA expression
3. Using MMT method to observe the cell proliferation condition.
4. Using TUNEL and flow cytometry methods to detect cell apoptosis situation.
5.Statistical analysis:Statistics analysis was used by SPSS13.0software, using chi-square test, t test and variance analysis and Spearmanm, test standard α=0.05.
Results:
1. The results of the method of MTT:Compared to the B group (Chkl siRNA2+cisplatin) and C group (cisplatin) and A group (normal control group), the cell growth inhibition rate increased dramatically, the differences were statistically significantly(P <0.05).
2. Immunocytochemistry and Western blot test results:B Group (Chkl siRNA2+cisplatin) and C group (cisplatin) and A group (normal control group), compared with EC9706esophageal cancer cells Chkl protein expression clear cut, two groups of difference in all have statistical significance (P<0.05).
3. The results of In situ hybridization and RT-PCR:the Chkl mRNA expressed in cells in group B(Chkl siRNA2+cisplatin) was significantly lower than the group C(cisplatin) and group A(normal control group), compared two groups of two differences were statistically significant (P<0.05).
4. Apoptosis results of TUNEL method:Cells in group B(Chkl siRNA2+cisplatin) apoptosis index (AI) is obviously higher than that of group C (cisplatin) and A group (normal control group), compared two groups of two differences were statistically significant (P<0.05).
5. Flow cell detection instrument apoptosis results:B Group (Chkl siRNA2+cisplatin) early apoptosis cells number and late apoptosis cells number than the C group (cisplatin) and A group (normal control group) increased considerably, and the differences were statistically significant (P<0.05).
Part Ⅳ The effect of Chk1siRNA interference expression vector on cisplatin induced esophageal carcinoma transplanted tumor cells biological behavior in vivo
Methods:
1. Cultivate esophageal cancer EC9706cell, constructing the nude mouse esophageal cancer transplantation tumor model.
2. Group breeding tumor-burdened nude mouse, namely A groups:normal group, pseudoscience injection Sterile Saline; B group:pseudoscience injection Chk1siRNA2add concentration for (5mg/kg) of cisplatin, C groups:pseudoscience injection cisplatin (5mg/kg). All group every3days one time, a total of5times.
3. Observe the transplantation tumor growth.
4. Using immunohistochemical, Western blot and in situ hybridization, RT polymerase chain reaction (PCR) to test the transplantation of Chkl protein and mRNA expression in the tumor tissues.
5.Using TUNEL method. to test the apoptosis situation of each cell.
6.Statistical analysis:Statistics analysis was used by SPSS13.0software, using chi-square test, T test and variance analysis and Spearmanm, test standard α=0.05.
Results:
1. Transplant pseudoscience product of the B group (Chkl siRNA2+cisplatin) noticeably less than the C group (cisplatin) and A group (normal control group), compared two groups of two, all have statistical difference (P<0.001).
2.The results of immunohistochemical and Western blot test:the expression of Chk1protein in the esophageal cancer transplantation tumor tissues of B group (Chk1siRNA2+cisplatin) was significantly lower than the C group (cisplatin) and A group (normal control group), compared two groups of two, all have statistical difference (P <0.05).
3.The results of in situ hybridization and RT-PCR detection:the expression of Chk1mRNA in the esophageal cancer transplantation tumor tissues of B group (Chk1 siRNA2+cisplatin) was significantly lower than the C group (cisplatin) and A group (normal control group), compared two groups of two, all have statistical difference (P <0.05).
4.The results of TUNEL method detection:the cells apoptosis index (AI) of group B(Chk1siRNA2+cisplatin) is obviously higher than that of the group C (cisplatin) and group A(normal control group), compared two groups of two, all have statistical difference (P<0.05).
Conclusion
1. High levels of Chkl protein and mRNA were observed in esophageal squamous cell carcinoma.
2. The expression of Chkl protein and mRNA were closely associated with the invasion and metastasis of ESCC.
3. Interfering expression vector pSilencer3.1-Chk1siRNA was successfully constructed.
4. Chk1siRNA2has the best inhibiting effect.
5. The expression of Chkl protein and mRNA can be cut by Chkl siRNA interference expression vector in vitro for people with esophageal cancer cells EC9706.
6. Silencing Chkl expression in vitro can enhance the esophageal cancer EC9706cell proliferation and induce its apoptosis sensitivity of which cisplatin on.
7. Successfully constructed nude mouse esophageal cancer transplantation tumor model.
8. Silencing Chkl expression can strengthen the body of nude mouse esophageal cancer transplantation tumor growth inhibition which cisplatin on and induce the effect of cell apoptosis of transplantation tumor.
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