香加皮有效成份抑制大鼠食管癌形成及其机制的实验研究
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摘要
食管癌是我国最常见的消化道恶性肿瘤之一,恶性程度高,发病原因不明,造成预防困难;因缺乏十分有效的诊治方法,发病率、死亡率居高不下,总5年生存率不超过10%。因此,通过预防降低食管癌发生率十分重要。流行病学调查资料显示,早期癌或癌前病变的发现、诊断和治疗是目前降低食管癌发病率和死亡率的主要措施,发现癌前病变逆转分化药物是降低食管癌死亡率的理想手段。中医药在肿瘤防治中的作用越来越受到人们的重视。已有大量研究证实,部分中药对食管癌的发生有一定防治作用,香加皮三萜类化合物(triterpenes compound of Cortex Periplocae,TCCP)是本实验室发现的既有抗肿瘤作用又有免疫调节活性的抗肿瘤活性成分,对胃癌、肝癌、乳腺癌都有较好的增殖抑制作用,对食管癌细胞的体外抑制效果也很明显,但是在体内抗肿瘤效果及作用机理尚不清楚。
本课题首先用甲基苄基亚硝胺(N-nitro-somethylbenzylamine, NMBA)诱导建立了大鼠食管癌前病变模型,检测了在食管癌形成过程中与细胞异常增殖相关的分子、蛋白及其基因表达的动态变化和相互关系,以及外周血免疫相关分子的变化,分析了香加皮三萜类化合物对NMBA诱发食管癌发生中上述分子表达变化的影响,为香加皮三萜类化合物的临床应用提供理论依据。
第一部分香加皮三萜类化合物对荷食管癌细胞裸鼠的抑瘤作用研究
目的:观察TCCP对荷食管鳞癌细胞裸鼠移植瘤的抑制作用,探讨其体内对荷食管癌移植瘤的抑制作用。
方法:将稳定表达荧光素酶基因(Luc)的人食管癌细胞株Eca109-luc细胞接种于裸鼠左侧肋腹部皮下,建立Eca109-luc细胞荷瘤裸鼠模型,待肿瘤生长至直径约5-7mm时,将裸鼠随机分为生理盐水处理组和TCCP(20mg/kg)治疗组,每组7只,隔日于瘤周注射生理盐水或药物一次,疗程4周。应用美国精诺真活体成像系统(Xenogen IVIS Imaging System)观察肿瘤生长情况,采用流式细胞仪分析肿瘤组织细胞的凋亡率;用RT-PCR和Western blot法检测肿瘤组织凋亡相关基因Fas和Survivin的表达变化。
结果:1经TCCP(20mg/kg)治疗荷Eca109-luc细胞裸鼠后,肿瘤的生长受到明显抑制,肿瘤体积明显小于盐水处理组(P<0.01);TCCP治疗组移植瘤的质量(0.82±0.14)g显著低于对照组(1.38±0.36)g(p<0.05),对肿瘤生长的抑制率为40.7%。
2经精诺真体内成像系统检测结果显示,TCCP治疗前裸鼠肿瘤光子数的平均值为5.47E+06±1.12E+06,与盐水处理组相比(5.02E+06±1.03E+06)无统计学差异(P>0.05);TCCP治疗后1、2、3、4周荷瘤裸鼠肿瘤的光子数分别为7.65E+06±2.14E+06、6.18E+07±1.36E+07、1.70E+08±1.14+08、5.53E+08±1.01E+08,均显著低于同时间点生理盐水处理组(6.48E+07±1.25E+07、3.65E+08±1.42E+08、7.64E+08±3.02E+07、3.34E+9±4.78E+08)(P均<0.01)。
3 FCM检测结果显示, TCCP治疗组裸鼠移植瘤细胞的凋亡率(32.5±1.2%)明显高于盐水处理组(11.6±1.0%),差异有显著性(P<0.05)。
4 RT-PCR分析结果显示,TCCP治疗组裸鼠瘤组织中Fas mRNA表达水平显著高于盐水处理组(P<0.01);而TCCP治疗组裸鼠瘤组织中Survivin mRNA的表达水平则显著低于盐水处理组(P<0.01)。
5 Western blot检测结果显示,TCCP治疗组荷瘤裸鼠瘤组织中Fas蛋白表达水平(0.725±0.046)明显高于盐水处理组(0.297±0.026)(P<0.01);而TCCP治疗组瘤组织中Survivin蛋白表达的水平(0.052±0.003)明显低于盐水对照组(0.387±0.016)(P<0.05)。
结论:TCCP在体内能明显抑制食管癌细胞增殖,其作用机制可能是调节Fas/FasL分子表达、下调肿瘤组织凋亡抑制基因Survivin的表达,从而促进肿瘤细胞的凋亡有关。
第二部分香加皮三萜类化合物对大鼠食管癌前病变的阻断作用及其机制研究
实验一大鼠食管癌前病变模型的建立及香加皮三萜类化合物对其阻断作用
目的:研究NMBA诱导F344大鼠食管上皮癌前病变形成过程中组织病理学改变、细胞周期调控因子和增殖相关蛋白表达的改变及TCCP对以上分子表达的影响。
方法:雄性F344大鼠165只,随机分为5组:1正常对照组(对照1组,n=15),常规饲养;2大豆油对照组(对照2组,n=15),每只大鼠注射大豆油1ml/kg,每周3次,共注射5周,常规饲养;3单纯诱癌组(n=45),皮下注射NMBA 0.5mg/kg,每周3次,共给药5周,常规饲养;4 TCCP高剂量干预组(干预1组,n=45),每只大鼠皮下注射NMBA(0.5mg/kg)后,同时肌注TCCP(20mg/kg),每周3次,共给药5周,常规饲养;5 TCCP低剂量干预组(干预2组,n=45),每只大鼠皮下注射NMBA(0.5mg/kg)后,同时肌注TCCP(10mg/kg),每周3次,共给药5周,常规饲养。在诱癌开始后9、15和25周时,各取对照1、2组大鼠5只,单纯诱癌组和干预1、2组各取15只大鼠,麻醉后解剖大鼠,肉眼观察食管粘膜大体变化;HE染色后光镜下观察食管上皮组织病理学变化;采用免疫组化方法检测各组食管组织PCNA、cyclinD1及CDK4蛋白表达变化。
结果:1实验动物一般状况:实验期间各组大鼠体重正常增长,各组间平均体重变化无显著性差异。实验后期单纯诱癌组大鼠出现皮毛蓬松,弓背卷缩现象,但体重仍在稳步上升,增长速度虽较治疗组稍慢,但无统计学意义(P>0.05)。
2各实验组食管标本大体观察:正常对照组及大豆油对照组在实验整个过程中未发现食管异常变化。NMBA诱癌大鼠食管出现以下变化:(1)食管粘膜增厚、发白及变粗糙。(2)粘膜表面出现点状、圆形、卵圆形或条状的白色斑块。这些斑块突起或平坦,直径一般0.1-0.2mm。在实验后期,白斑病变增多、变大与融合。(3)形成乳头状瘤,一般单发或多发,有时散布在食管各段。乳头状瘤呈球形、卵圆形、半球形或丘状突起,表面呈莱花状,直径约1mm。单纯诱癌组大鼠在诱癌开始后9周大鼠食管上皮仅见粘膜增厚和白斑形成,15周时有20%(3/15)的大鼠出现单发的乳头状瘤样病变(以大于1 mm计算),TCCP高剂量和低剂量干预组均未见乳头状瘤样病变发生;25周时,60.0%(6/15)单纯诱癌组大鼠食管可见单发或多发乳头状瘤样病变,平均每只大鼠的瘤数为0.93,TCCP高剂量干预组和低剂量干预组大鼠的乳头状瘤发生率分别降低至20%(P<0.01)和33.3%(P>0.05),平均每只大鼠瘤数也分别下降至0.27和0.33。两干预组相比无统计学差异(P>0.05)。
3各实验组食管标本组织病理学观察:用NMBA诱癌开始后9周时,单纯诱癌组大鼠癌前病变发生率为20.0%,15周时46.7%,至25周时达93.3%。与单纯诱癌组相比,诱癌9、15周时TCCP高剂量和低剂量药物干预组食管上皮未发生癌前病变大鼠比率明显升高(P<0.05)。25周时,与单纯诱癌组相比,高剂量和低剂量TCCP对模型大鼠癌前病变发生的抑制率分别达64.3%和50%(P<0.05)。
4诱癌开始后9、15和25周时,单纯诱癌组大鼠食管上皮组织PCNA表达水平分别为213.17±29.74、268.35±39.56、327.24±28.19,均显著高于正常对照组的表达水平(167.96±20.16、170.76±14.79、172.49±17.49)(P均<0.05);与单纯诱癌组相比,TCCP高剂量和低剂干预量组大鼠食管上皮组织中PCNA表达水平均显著降低(P<0.05)。
5正常对照组和大豆油对照组大鼠食管上皮组织CDK4和cyclinD1呈阴性表达。单纯诱癌组大鼠食管组织中随用NMBA诱癌时间的延长CDK4和cyclinD1的表达水平逐渐增强,两者的阳性表达变化呈明显相关性(r=0.9631, P<0.05)。与单纯诱癌组相比,高、低剂量TCCP干预组在诱癌后9、15及25周时,大鼠食管上皮组织中CDK4和cyclinD1表达水平均明显降低(P<0.05);在诱癌9周时,高剂量与低剂量组相比,大鼠食管上皮组织CDK4和cyclinD1的表达水平无显著性差异;诱癌15周和25周时,高剂量组食管组织CDK4和cyclinD1阳性表达水平明显低于低剂量组(P<0.05)。
结论:甲基苄基亚硝胺(0.5mg/kg)可诱导F344大鼠食管癌前病变的发生;单纯诱癌组大鼠食管上皮出现典型的癌前病变组织病理学变化,食管上皮组织中增殖相关蛋白PCNA及周期调控因子CDK4和cyclinD1的表达水平明显升高;香加皮三萜类化合物早期干预可明显抑制甲基苄基亚硝胺诱导大鼠食管癌前病变的发生与发展,该作用可能与其抑制大鼠食管组织上皮中PCNA、CDK4和cyclinD1表达有关。
实验二香加皮三萜类化合物对大鼠食管癌前病变形成过程中Wnt信号通路的影响
目的:研究TCCP对NMBA诱导F344大鼠食管癌前病变形成过程中,Wnt信号通路分子表达变化,进一步探讨其抑制食管癌前病变的作用机理。
方法:分别取各组大鼠食管组织,用RT-PCR法检测c-myc mRNA表达水平;用Western blot法检测GSK-3β和β-catenin蛋白表达水平。
结果:1与正常对照组相比,用NMBA诱癌9周时,大鼠食管上皮组织中c-myc mRNA表达水平显著升高,且随诱癌时间的延长表达水平逐渐升高,至诱癌25周时达峰值。与单纯诱癌组相比,在诱癌9、15周时高低剂量TCCP干预组大鼠食管上皮组织中c-myc mRNA表达水平均显著下降,且高剂量组c-myc mRNA的表达水平明显低于低剂量组(P<0.05)。诱癌25周时,两剂量TCCP干预组食管上皮组织c-myc mRNA表达水平虽较单纯诱癌组降低,但无统计学意义(P>0.05)。2正常对照组及大豆油对照组大鼠食管组织有β-catenin蛋白表达;在NMBA诱癌9周时大鼠食管上皮组织中β-catenin的表达水平明显升高,与正常对照组相比差异显著(P<0.05)。随诱癌时间延长,其表达水平逐渐升高。与正常对照及大豆油对照组大鼠相比,在诱癌9周时单纯诱癌组大鼠食管组织GSK-3β的蛋白表达水平显著下降,随着诱癌时间延长表达水平逐渐降低,至25周达最低水平(P<0.05)。
在诱癌后9、15和25周时,与单纯诱癌组相比,高、低剂量TCCP干预组大鼠食管组织β-catenin蛋白表达水平均显著下降(P<0.05),而GSK3β蛋白表达显著升高(P<0.05);高剂量TCCP组大鼠食管组织β-catenin及GSK3β蛋白表达水平,与低剂量组相比无明显差异(P>0.05)。
结论:TCCP可能通过促进Wnt信号分子GSK-3β的表达,抑制β-catenin蛋白的表达,进而下调下游靶基因c-myc的转录,干扰细胞周期转化,逆转细胞的分化,可能是其抑制食管癌变细胞生长机制之一。
第三部分香加皮三萜类化合物对食管癌前病变大鼠免疫调节作用的实验研究
目的:探讨TCCP抑制大鼠食管癌前病变形成过程中免疫调节作用机理。
方法:1采用流式细胞技术分析TCCP对NMBA诱癌大鼠外周血中T细胞亚群CD4+CD25+调节性T细胞比率的影响,用半定量RT-PCR技术分析其对大鼠外周血单个核细胞(PBMC)中Foxp3 mRNA表达水平的影响。
2应用RT-PCR技术分析香TCCP对NMBA诱癌大鼠外周血PBMC中细胞因子IL-2、IL-10、TGF-βmRNA表达水平的影响,应用ELISA检测技术分析TCCP对NMBA诱癌大鼠外周血中细胞因子IL-2、IL-10、TGF-β水平的影响。
结果:1与正常对照组比,用NMBA诱癌9周时,单纯诱癌组外周血中CD4+T细胞比例显著降低,CD4+CD25+Treg细胞比例及Foxp3 mRNA表达水平明显升高,此现象随诱癌时间的延长而加重(P<0.05);在诱癌9周、15周时,与单纯诱癌组相比,高、低剂量TCCP干预组大鼠外周血中CD4+T细胞比例明显升高,CD4+CD25+Treg细胞比例及Foxp3 mRNA表达水平明显降低(P<0.05);诱癌25周时,与单纯诱癌组相比,两剂量TCCP干预组显著降低了Foxp3 mRNA的表达水平,但对CD4+CD25+Treg细胞和CD4+T细胞的比率无明显影响(P>0.05)。高剂量TCCP组大鼠外周血以上指标的水平与低剂量组相比无明显差异(P>0.05)。
2与正常对照组相比,用NMBA诱癌9周时,单纯诱癌组大鼠外周血单个核细胞中IL-10、TGF-βmRNA表达水平及其血清中相应蛋白含量显著升高,而IL-2 mRNA表达水平及其血清中相应蛋白含量显著下降(P<0.05),15周时各指标水平虽有所升高,但与9周比无显著性差异(P>0.05);与15周相比,至25周时,IL-10、TGF-βmRNA表达水平及其血清中相应蛋白含量显著升高,而IL-2 mRNA表达水平及其血清中相应蛋白含量显著下降(P<0.05)。与单纯诱癌组相比,用NMBA诱癌9、15周时,高、低剂量TCCP干预组大鼠外周血单个核细胞中IL-10、TGF-βmRNA表达水平及其血清中相应蛋白含量显著下降(P<0.05),IL-2 mRNA表达水平及其血清中相应蛋白含量显著升高(P<0.05);25周时,高、低剂量TCCP干预组对以上细胞因子基因表达及蛋白含量无明显影响(P>0.05)。
结论:TCCP可抑制NMBA诱导大鼠癌前病变形成的免疫调节作用,其作用机制可能与其下调外周血CD4+CD25+Tr细胞比例及Foxp3 mRNA表达水平有关;可能与其抑制大鼠免疫系统产生Th2类细胞因子,促进Th1类细胞因子有关。
Esophageal carcinoma is a kind of most common malignant tumor in China with high degree of malignity. Despite the persistent development of treatment methods in recent years, the long-term follow-up result of esophageal carcinoma, on a whole, is still unsatisfied and the prognosis is very inclement with high morbility and mortality (overall 5-year survival rates less than 10%). So preventive measure may be the best way to reduce the incidence of esophageal carcinoma in current. Epidemiological data showed that early recognition, diagnosis and treatment of cancer or preneoplastic lesions is the main measure to reduce the mortality of esophageal cancer, and seeking the drug which can reverse the development of prenoplastic lesions appears to be a useful method. In recent years, the effect of traditional Chinese medicine on cancer prevention has aroused more and more intreasts and many studies have proved that some Chinese herbs may prevent esophageal cancer. Triterpenes compound of Cortex Periplocae (TCCP) is an active component of Cortex Periplocae with anti-tumor and immunoregulation activity which is developed by our laboratory. TCCP has the significant inhibitory effect on tumor proliferation such as stomach cancer, hepatocarcinoma, or breast cancer as well as esophageal carcinoma cells in vitro. However, a further research need to be developed for whether it can reverse the development of prenoplastic lesion of esophagous in vivo.
In this present study, we treated F344 rats with the esophageal carcinogen, N-nitrosomethylbenzylamine (NMBA), thrice per week for 5 weeks to establish a model of human esophageal squamous cell carcinoma. Histochemistry, immunohistochemistry, RT-PCR and western blot technique were used in this study to investigate the variable trends of expression and correlation of some proteins and genes interrelated with abnormal proliferation of esophageal epithelium cell during periods of esophageal tumorigenesis induced by NMBA and the effect of TCCP. The purpose of the study is to screen out an applicable and effective esophageal carcinoma preventive agent of traditional Chinese medicine, to offer academic foundation for further clinic study and to spread out its application.
Part 1 The observation of tumor-inhibitory effect of TCCP on the nude mice bearing esophageal carcinoma cells
Objective: To observe the inhibitory effect of TCCP on the nude mice bearing esophageal squmous carcinoma cells and explore the inhibitory mechanism in vivo.
Methods: Eca109-luc stablely expressing luciferase were inoculated subcutaneously into the hind flank region of nude mice to establish tumor model. Nude mice were randomly grouped until tuomr’s length reached 5-7 mm. One group of mice (n=7) was treated i.s. with NS. A second group of mice (n=7) was treated i.s with TCCP (20mg/kg) thrice per week. The course of all the treatments would last for 4 weeks. Tumor growth were monitored with in vivo imaging system. Cell apoptosis in vivo were analyzed by flow cytometry. Western blot was used to investigate protein expression of Survivin.
Results: 1 Compared with NS control group, the tumor growth of the nude mice bearing Eca109-luc was significantly inhibited by TCCP treatment (1.38±0.36 g vs 0.82±0.14 g), with the inhibitory ratio 40.7%.
2 There was no significant difference in photon number detected by in vivo bioluminescence imaging between NS control group and TCCP group before treatment with TCCP (5.02E+06±1.03E+06, 5.47E+06±1.12E+06 respectively)( P>0.05). After treatment with TCCP, the average photon number of the TCCP group were decreased by 7.65E+06±2.14E+06、6.18E+07±1.36E+07、1.70E+08±1.14+08 and 5.53E+08±1.01E+08 at weeks 1, 2, 3 and 4, respectively, significantly lower than NS control group (6.48E+07±1.25E+07、3.65E+08±1.42E+08、7.64E+08±3.02E+07 and 3.34E+9±4.78E+08, respectively) (P<0.01).
3 FCM assay for the apoptosis rate of tumor cells in vivo showed that there was a significant difference between the NS control group (11.6±1.0%) and the TCCP group (32.5±1.2%) (P<0.05).
4 Gene expression of Fas in TCCP treated mice was significantly higher than that of NS group(P<0.01), while survivin mRNA was significantly decreased.
5 It was observed that the protein expreesion of Fas in the tumor tissues of TCCP group was significantly higher (0.725±0.046), as compared with that of NS control group (0.297±0.026)(P<0.01), while the protein expreesion of Survivin was significantly decreased (0.052±0.003), as compared with that of NS control group (0.387±0.016)(P<0.05).
Conclusions: Cell growth in vivo was inhibited significantly by TCCP which might exert its anti-tumor activity through Fas/FasL pathway related to reduction of survivin expression, and then inducing apoptosis of tumor cells.
Part 2 Effect of TCCP on NMBA-induced rats esophageal carcinogenesis and its mechanism
1 The establish of rat model of esophageal preneoplastic lesion and the effect of TCCP on the cacinogenesis
Objective: To investigate the effect of TCCP on esophageal pathology , proliferating cell muclear antigen (PCNA) expression and cell cycle regulatory components in NMBA-induced rat tumorigenesis.
Methods: 165 male F344 rats (5-6 weeks of age) were randomly divided into five experimental groups according to the different regiments. It consisted of the following groups: (1) normal control groups (group 1, n=15): rats were raised rountinly; (2) soya oil control groups (group 2, n=15): rats were injected with soya oil 1ml/kg intramuscularily (i.m.); (3) NMBA control groups (group 3, n= 45): rats were injected with NMBA 0.5mg/kg only; (4) Low dose TCCP treatment groups (group 4, n=45): rats received NMBA 0.5mg/kg s.c. plus TCCP 20mg/kg i.m.; (5) High dose TCCP treatment groups (group 5, n=45): rats received NMBA 0.5mg/kg s.c. plus TCCP 10mg/kg i.m..The administration of drugs was scheduled as below: thrice per week for 5 weeks. At 9, 15 and 25 week, 5 rats from groups 1 and 2 and 15 rats from groups 3, 4 and 5 were euthanized by pentobarbital sodium and subjected to gross necropsy. The esophagus of each rat was excised and opened longitudinally. Then esophagus were fixed in 10% phosphate-buffer formalin solution, and routinely embedded in paraffin for HE staining to observe the pathological changes of esophageal tissues, while the expression of PCNA, cyclinD1 and CDK4 was measured by immunohistochemistry.
Results: 1 General observations. The mean body weights and food consumption in all rats were not significantly different throughout the bioassay. At the end of the bioassay rats treated with NMBA were found in low spirits with shaggy fur. However, body weights still increased.
2 General observations of esophagi of rats. There were no abnormal changes in normal and soya oil group among bioassay. Changes were observed in NMBA-induced rats as following: (1) esophageal mucosa becoming incrassation with color white and scabrities; (2) white patch. there were some pathologic white patch changes on the surface of esophageal mucosa just like dot, circle, orbicular-ovate or strip with size about 0.1-0.2 mm and the area of the pathologic changes increased at the end of bioassay. (3) papilloma. At week 9 of the bioassay, esophagi had pathologic changes like incrassation and white patch. At weeks 15 and 25 of the bioassay, 20% to 60% of the esophagi, respectively, had papillomas. Mean number of papillomas per rat is 0.93 at week 25.TCCP showed significant inhibitory effects on the incidence of the papilloma in group 4 to 20% ( P<0.01), and the mean number of papillomas was also decreased to 0.27. In group 5, the decrease of incidence and number of the papilloma was not statistically significant (33.3%, 0.33, respectively). There was also no statistically significant between group 4 and group 5.
3 The results of histopathologic examination of esophagi. The preneoplatic lesion of esophageal tissues was evaluated. At weeks 9, 15 and 25, the incidence of preneoplastic lesion in groups 3 was 20.0%, 46.7% and 93.3 %, respectively. TCCP markedly inhibited rat esophageal preneoplastic lesion, the inhibitory rates at 25 weeks were 64.3% in group 4 and 50% in group 5, respectively (P<0.05).
4 There were a markedly increase in the expression of PCNA in NMBA control group (week 9: 213.17±29.74; week 15: 268.35±39.56; week 25: 327.24±28.19), as compared with that in normal control group(167.96±20.16、170.76±14.79、172.49±17.49, respectively)( P <0.05). TCCP significantly decreased the expression of PCNA in groups 4 (185.28±22.98、200.56±28.19、282.23±37.43) and group 5 (189.27±22.10、209.73±28.88、305.60±17.46) (P<0.05).
5 The expreesion of CDK4 and cyclin D1 were not found in normal mucosa of esophagi of rats in normal control and soya oil control group, but were induced by administering NMBA. Positive expression of cyclinD1 and CDK4 increased gradually and reached the peak at 25wk. There were significant positive correlations between the two protein expression (r=0.9631, P<0.05). Treatment with TCCP could significantly decrease the protein expression of CDK4 at weeks 9, 15 and 25 (P<0.05). And there was significant difference between the high TCCP group and low TCCP group at weeks 15 and 25. TCCP could act a similar effect on the expreesion of cyclinD1.
Conclusions: NMBA (0.5mg/kg) can sucessfully induced F344 rats model of esophageal preneoplastic lesion. Typical histophathologic changes was examined in the NMBA-treated rats. TCCP may restrain the high expression of PCNA, while alleviate the esophageal preneoplastic lesion of rats induced by NMBA. TCCP can also inhibit the uncontrolled proliferation of esophageal epithelia cells and regulating cell-cycle process.
2 The effect of TCCP on wnt signal pathway in NMBA-induced rat tumorigenesis
Objective: To study the effect of TCCP on wnt signal pathway in NMBA-induced rats tumorigenesis and to investigate the mechanism by which TCCP modulate tumorigenesis.
Methods: Total cellular RNA was isolated from frozen tissues using TRIzol Reagent. The expression of c-myc mRNA was detected by RT-PCR. Western blot was used to investigate the protein expression of GSK-3βandβ-catenin in the esophageal epithelium.
Results: 1 The gene expression of c-myc of esophageal epithelium in NMBA control group was significantly increased at weeks 9, 15, 25 compared with normal control (P<0.05). TCCP supressed the mRNA expression of c-myc at both weeks 9 and 15 (P<0.05), but not at weeks 25.There was no significant difference between the high TCCP group and low TCCP group.
2 It was observed that the expression ofβ-catenin was slightly present in normal mucosa of esophagi of the rats which were untreated with NMBA, and then was significantly increased by administering NMBA from weeks 9 to 25 (P<0.05). The up-regulatedβ-catenin expression was decreased significantly by TCCP treatment at each check-point (P<0.05). The expression of GSK-3βwas rich in normal mucoa of esophagus, but was decreased significantly by administering NMBA, and was lowest at 25th week (P<0.05). The decreased protein levels of GSK3βwas significantly elevated by TCCP treatment at each check-point (P<0.05). But there was no significantly between the two treatment groups.
Conclusions: TCCP inhibited NMBA-induced rat esophageal carcinogenesis probably via acitivition of GSK-3β, suppression ofβ-catenin and c-myc expression.
Part 3 Effects of triterpenes compound of Cortex Periplocae on cyto-immunologic function in N-nitrosomethylbenzylamine-induced esophageal tumorigenesis in F344 rats
Objective: To investigate the mechanism and effects of triterpenes compound of Cortex Periplocae on cyto-immunologic function in N-nitrosomethyl- benzylamine-induced esophageal tumorigenesis in F344 rats.
Methods: 1 FCM and RT-PCR were used to detected the proportion of CD4+CD25+ and gene expression of Foxp3 in peripheral blood of rats . 2 RT-PCR and ELASA were used to detected the gene and protein expression of IL-2, IL-10 and TGF-β1 in peripheral blood of rats .
Results: 1 At week 9 after the NMBA treatment, CD4+T cells in peripheral blood of NMBA group rats were decreased significantly and CD4+CD25+ regulatory T cells and Foxp3 mRNA in peripheral blood of NMBA group rats were significantly increased, which deteriorated further with the time going on (P<0.05). At weeks 9 and 15, TCCP treatment can decreased the proportions of CD4+CD25+ regulatory T cells and the gene expression of Foxp3, while the proportions of CD4+ cells were significantly increased (P<0.05), compared with NMBA group. At week 25, TCCP significantly decreased the expression of Foxp3 mRNA, but had no significant effect on CD4+CD25+Treg and CD4+T cell.
At week 9 after NMBA treatment, the protein levels of TGF-β1 and IL-10 in peripheral blood of in F344 rats were significantly higher than those in normal control group and soy control group. The gene expression of TGF-β1 and IL-10 were also significantly elevated. While the levels of protein and gene expression of IL-2 were significantly decreased, as compared with normal control. TCCP can decrease the expression of IL-10 and TGF-β1, increase the expression of IL-2.
Conclusions: The expression of CD4+CD25+ regulatory T cells and Foxp3 mRNA in peripheral blood of rats were enhanced after treatment with NMBA, while the triterpenes compound of Cortex Periplocae can effectively inhibit the expression, thus it obviously improve the immune dysfunction in experimental esophageal tumorigenesis in F344 rats.
本课题首先用甲基苄基亚硝胺(N-nitro-somethylbenzylamine, NMBA)诱导建立了大鼠食管癌前病变模型,检测了在食管癌形成过程中与细胞异常增殖相关的分子、蛋白及其基因表达的动态变化和相互关系,以及外周血免疫相关分子的变化,分析了香加皮三萜类化合物对NMBA诱发食管癌发生中上述分子表达变化的影响,为香加皮三萜类化合物的临床应用提供理论依据。
第一部分香加皮三萜类化合物对荷食管癌细胞裸鼠的抑瘤作用研究
目的:观察TCCP对荷食管鳞癌细胞裸鼠移植瘤的抑制作用,探讨其体内对荷食管癌移植瘤的抑制作用。
方法:将稳定表达荧光素酶基因(Luc)的人食管癌细胞株Eca109-luc细胞接种于裸鼠左侧肋腹部皮下,建立Eca109-luc细胞荷瘤裸鼠模型,待肿瘤生长至直径约5-7mm时,将裸鼠随机分为生理盐水处理组和TCCP(20mg/kg)治疗组,每组7只,隔日于瘤周注射生理盐水或药物一次,疗程4周。应用美国精诺真活体成像系统(Xenogen IVIS Imaging System)观察肿瘤生长情况,采用流式细胞仪分析肿瘤组织细胞的凋亡率;用RT-PCR和Western blot法检测肿瘤组织凋亡相关基因Fas和Survivin的表达变化。
结果:1经TCCP(20mg/kg)治疗荷Eca109-luc细胞裸鼠后,肿瘤的生长受到明显抑制,肿瘤体积明显小于盐水处理组(P<0.01);TCCP治疗组移植瘤的质量(0.82±0.14)g显著低于对照组(1.38±0.36)g(p<0.05),对肿瘤生长的抑制率为40.7%。
2经精诺真体内成像系统检测结果显示,TCCP治疗前裸鼠肿瘤光子数的平均值为5.47E+06±1.12E+06,与盐水处理组相比(5.02E+06±1.03E+06)无统计学差异(P>0.05);TCCP治疗后1、2、3、4周荷瘤裸鼠肿瘤的光子数分别为7.65E+06±2.14E+06、6.18E+07±1.36E+07、1.70E+08±1.14+08、5.53E+08±1.01E+08,均显著低于同时间点生理盐水处理组(6.48E+07±1.25E+07、3.65E+08±1.42E+08、7.64E+08±3.02E+07、3.34E+9±4.78E+08)(P均<0.01)。
3 FCM检测结果显示, TCCP治疗组裸鼠移植瘤细胞的凋亡率(32.5±1.2%)明显高于盐水处理组(11.6±1.0%),差异有显著性(P<0.05)。
4 RT-PCR分析结果显示,TCCP治疗组裸鼠瘤组织中Fas mRNA表达水平显著高于盐水处理组(P<0.01);而TCCP治疗组裸鼠瘤组织中Survivin mRNA的表达水平则显著低于盐水处理组(P<0.01)。
5 Western blot检测结果显示,TCCP治疗组荷瘤裸鼠瘤组织中Fas蛋白表达水平(0.725±0.046)明显高于盐水处理组(0.297±0.026)(P<0.01);而TCCP治疗组瘤组织中Survivin蛋白表达的水平(0.052±0.003)明显低于盐水对照组(0.387±0.016)(P<0.05)。
结论:TCCP在体内能明显抑制食管癌细胞增殖,其作用机制可能是调节Fas/FasL分子表达、下调肿瘤组织凋亡抑制基因Survivin的表达,从而促进肿瘤细胞的凋亡有关。
第二部分香加皮三萜类化合物对大鼠食管癌前病变的阻断作用及其机制研究
实验一大鼠食管癌前病变模型的建立及香加皮三萜类化合物对其阻断作用
目的:研究NMBA诱导F344大鼠食管上皮癌前病变形成过程中组织病理学改变、细胞周期调控因子和增殖相关蛋白表达的改变及TCCP对以上分子表达的影响。
方法:雄性F344大鼠165只,随机分为5组:1正常对照组(对照1组,n=15),常规饲养;2大豆油对照组(对照2组,n=15),每只大鼠注射大豆油1ml/kg,每周3次,共注射5周,常规饲养;3单纯诱癌组(n=45),皮下注射NMBA 0.5mg/kg,每周3次,共给药5周,常规饲养;4 TCCP高剂量干预组(干预1组,n=45),每只大鼠皮下注射NMBA(0.5mg/kg)后,同时肌注TCCP(20mg/kg),每周3次,共给药5周,常规饲养;5 TCCP低剂量干预组(干预2组,n=45),每只大鼠皮下注射NMBA(0.5mg/kg)后,同时肌注TCCP(10mg/kg),每周3次,共给药5周,常规饲养。在诱癌开始后9、15和25周时,各取对照1、2组大鼠5只,单纯诱癌组和干预1、2组各取15只大鼠,麻醉后解剖大鼠,肉眼观察食管粘膜大体变化;HE染色后光镜下观察食管上皮组织病理学变化;采用免疫组化方法检测各组食管组织PCNA、cyclinD1及CDK4蛋白表达变化。
结果:1实验动物一般状况:实验期间各组大鼠体重正常增长,各组间平均体重变化无显著性差异。实验后期单纯诱癌组大鼠出现皮毛蓬松,弓背卷缩现象,但体重仍在稳步上升,增长速度虽较治疗组稍慢,但无统计学意义(P>0.05)。
2各实验组食管标本大体观察:正常对照组及大豆油对照组在实验整个过程中未发现食管异常变化。NMBA诱癌大鼠食管出现以下变化:(1)食管粘膜增厚、发白及变粗糙。(2)粘膜表面出现点状、圆形、卵圆形或条状的白色斑块。这些斑块突起或平坦,直径一般0.1-0.2mm。在实验后期,白斑病变增多、变大与融合。(3)形成乳头状瘤,一般单发或多发,有时散布在食管各段。乳头状瘤呈球形、卵圆形、半球形或丘状突起,表面呈莱花状,直径约1mm。单纯诱癌组大鼠在诱癌开始后9周大鼠食管上皮仅见粘膜增厚和白斑形成,15周时有20%(3/15)的大鼠出现单发的乳头状瘤样病变(以大于1 mm计算),TCCP高剂量和低剂量干预组均未见乳头状瘤样病变发生;25周时,60.0%(6/15)单纯诱癌组大鼠食管可见单发或多发乳头状瘤样病变,平均每只大鼠的瘤数为0.93,TCCP高剂量干预组和低剂量干预组大鼠的乳头状瘤发生率分别降低至20%(P<0.01)和33.3%(P>0.05),平均每只大鼠瘤数也分别下降至0.27和0.33。两干预组相比无统计学差异(P>0.05)。
3各实验组食管标本组织病理学观察:用NMBA诱癌开始后9周时,单纯诱癌组大鼠癌前病变发生率为20.0%,15周时46.7%,至25周时达93.3%。与单纯诱癌组相比,诱癌9、15周时TCCP高剂量和低剂量药物干预组食管上皮未发生癌前病变大鼠比率明显升高(P<0.05)。25周时,与单纯诱癌组相比,高剂量和低剂量TCCP对模型大鼠癌前病变发生的抑制率分别达64.3%和50%(P<0.05)。
4诱癌开始后9、15和25周时,单纯诱癌组大鼠食管上皮组织PCNA表达水平分别为213.17±29.74、268.35±39.56、327.24±28.19,均显著高于正常对照组的表达水平(167.96±20.16、170.76±14.79、172.49±17.49)(P均<0.05);与单纯诱癌组相比,TCCP高剂量和低剂干预量组大鼠食管上皮组织中PCNA表达水平均显著降低(P<0.05)。
5正常对照组和大豆油对照组大鼠食管上皮组织CDK4和cyclinD1呈阴性表达。单纯诱癌组大鼠食管组织中随用NMBA诱癌时间的延长CDK4和cyclinD1的表达水平逐渐增强,两者的阳性表达变化呈明显相关性(r=0.9631, P<0.05)。与单纯诱癌组相比,高、低剂量TCCP干预组在诱癌后9、15及25周时,大鼠食管上皮组织中CDK4和cyclinD1表达水平均明显降低(P<0.05);在诱癌9周时,高剂量与低剂量组相比,大鼠食管上皮组织CDK4和cyclinD1的表达水平无显著性差异;诱癌15周和25周时,高剂量组食管组织CDK4和cyclinD1阳性表达水平明显低于低剂量组(P<0.05)。
结论:甲基苄基亚硝胺(0.5mg/kg)可诱导F344大鼠食管癌前病变的发生;单纯诱癌组大鼠食管上皮出现典型的癌前病变组织病理学变化,食管上皮组织中增殖相关蛋白PCNA及周期调控因子CDK4和cyclinD1的表达水平明显升高;香加皮三萜类化合物早期干预可明显抑制甲基苄基亚硝胺诱导大鼠食管癌前病变的发生与发展,该作用可能与其抑制大鼠食管组织上皮中PCNA、CDK4和cyclinD1表达有关。
实验二香加皮三萜类化合物对大鼠食管癌前病变形成过程中Wnt信号通路的影响
目的:研究TCCP对NMBA诱导F344大鼠食管癌前病变形成过程中,Wnt信号通路分子表达变化,进一步探讨其抑制食管癌前病变的作用机理。
方法:分别取各组大鼠食管组织,用RT-PCR法检测c-myc mRNA表达水平;用Western blot法检测GSK-3β和β-catenin蛋白表达水平。
结果:1与正常对照组相比,用NMBA诱癌9周时,大鼠食管上皮组织中c-myc mRNA表达水平显著升高,且随诱癌时间的延长表达水平逐渐升高,至诱癌25周时达峰值。与单纯诱癌组相比,在诱癌9、15周时高低剂量TCCP干预组大鼠食管上皮组织中c-myc mRNA表达水平均显著下降,且高剂量组c-myc mRNA的表达水平明显低于低剂量组(P<0.05)。诱癌25周时,两剂量TCCP干预组食管上皮组织c-myc mRNA表达水平虽较单纯诱癌组降低,但无统计学意义(P>0.05)。2正常对照组及大豆油对照组大鼠食管组织有β-catenin蛋白表达;在NMBA诱癌9周时大鼠食管上皮组织中β-catenin的表达水平明显升高,与正常对照组相比差异显著(P<0.05)。随诱癌时间延长,其表达水平逐渐升高。与正常对照及大豆油对照组大鼠相比,在诱癌9周时单纯诱癌组大鼠食管组织GSK-3β的蛋白表达水平显著下降,随着诱癌时间延长表达水平逐渐降低,至25周达最低水平(P<0.05)。
在诱癌后9、15和25周时,与单纯诱癌组相比,高、低剂量TCCP干预组大鼠食管组织β-catenin蛋白表达水平均显著下降(P<0.05),而GSK3β蛋白表达显著升高(P<0.05);高剂量TCCP组大鼠食管组织β-catenin及GSK3β蛋白表达水平,与低剂量组相比无明显差异(P>0.05)。
结论:TCCP可能通过促进Wnt信号分子GSK-3β的表达,抑制β-catenin蛋白的表达,进而下调下游靶基因c-myc的转录,干扰细胞周期转化,逆转细胞的分化,可能是其抑制食管癌变细胞生长机制之一。
第三部分香加皮三萜类化合物对食管癌前病变大鼠免疫调节作用的实验研究
目的:探讨TCCP抑制大鼠食管癌前病变形成过程中免疫调节作用机理。
方法:1采用流式细胞技术分析TCCP对NMBA诱癌大鼠外周血中T细胞亚群CD4+CD25+调节性T细胞比率的影响,用半定量RT-PCR技术分析其对大鼠外周血单个核细胞(PBMC)中Foxp3 mRNA表达水平的影响。
2应用RT-PCR技术分析香TCCP对NMBA诱癌大鼠外周血PBMC中细胞因子IL-2、IL-10、TGF-βmRNA表达水平的影响,应用ELISA检测技术分析TCCP对NMBA诱癌大鼠外周血中细胞因子IL-2、IL-10、TGF-β水平的影响。
结果:1与正常对照组比,用NMBA诱癌9周时,单纯诱癌组外周血中CD4+T细胞比例显著降低,CD4+CD25+Treg细胞比例及Foxp3 mRNA表达水平明显升高,此现象随诱癌时间的延长而加重(P<0.05);在诱癌9周、15周时,与单纯诱癌组相比,高、低剂量TCCP干预组大鼠外周血中CD4+T细胞比例明显升高,CD4+CD25+Treg细胞比例及Foxp3 mRNA表达水平明显降低(P<0.05);诱癌25周时,与单纯诱癌组相比,两剂量TCCP干预组显著降低了Foxp3 mRNA的表达水平,但对CD4+CD25+Treg细胞和CD4+T细胞的比率无明显影响(P>0.05)。高剂量TCCP组大鼠外周血以上指标的水平与低剂量组相比无明显差异(P>0.05)。
2与正常对照组相比,用NMBA诱癌9周时,单纯诱癌组大鼠外周血单个核细胞中IL-10、TGF-βmRNA表达水平及其血清中相应蛋白含量显著升高,而IL-2 mRNA表达水平及其血清中相应蛋白含量显著下降(P<0.05),15周时各指标水平虽有所升高,但与9周比无显著性差异(P>0.05);与15周相比,至25周时,IL-10、TGF-βmRNA表达水平及其血清中相应蛋白含量显著升高,而IL-2 mRNA表达水平及其血清中相应蛋白含量显著下降(P<0.05)。与单纯诱癌组相比,用NMBA诱癌9、15周时,高、低剂量TCCP干预组大鼠外周血单个核细胞中IL-10、TGF-βmRNA表达水平及其血清中相应蛋白含量显著下降(P<0.05),IL-2 mRNA表达水平及其血清中相应蛋白含量显著升高(P<0.05);25周时,高、低剂量TCCP干预组对以上细胞因子基因表达及蛋白含量无明显影响(P>0.05)。
结论:TCCP可抑制NMBA诱导大鼠癌前病变形成的免疫调节作用,其作用机制可能与其下调外周血CD4+CD25+Tr细胞比例及Foxp3 mRNA表达水平有关;可能与其抑制大鼠免疫系统产生Th2类细胞因子,促进Th1类细胞因子有关。
Esophageal carcinoma is a kind of most common malignant tumor in China with high degree of malignity. Despite the persistent development of treatment methods in recent years, the long-term follow-up result of esophageal carcinoma, on a whole, is still unsatisfied and the prognosis is very inclement with high morbility and mortality (overall 5-year survival rates less than 10%). So preventive measure may be the best way to reduce the incidence of esophageal carcinoma in current. Epidemiological data showed that early recognition, diagnosis and treatment of cancer or preneoplastic lesions is the main measure to reduce the mortality of esophageal cancer, and seeking the drug which can reverse the development of prenoplastic lesions appears to be a useful method. In recent years, the effect of traditional Chinese medicine on cancer prevention has aroused more and more intreasts and many studies have proved that some Chinese herbs may prevent esophageal cancer. Triterpenes compound of Cortex Periplocae (TCCP) is an active component of Cortex Periplocae with anti-tumor and immunoregulation activity which is developed by our laboratory. TCCP has the significant inhibitory effect on tumor proliferation such as stomach cancer, hepatocarcinoma, or breast cancer as well as esophageal carcinoma cells in vitro. However, a further research need to be developed for whether it can reverse the development of prenoplastic lesion of esophagous in vivo.
In this present study, we treated F344 rats with the esophageal carcinogen, N-nitrosomethylbenzylamine (NMBA), thrice per week for 5 weeks to establish a model of human esophageal squamous cell carcinoma. Histochemistry, immunohistochemistry, RT-PCR and western blot technique were used in this study to investigate the variable trends of expression and correlation of some proteins and genes interrelated with abnormal proliferation of esophageal epithelium cell during periods of esophageal tumorigenesis induced by NMBA and the effect of TCCP. The purpose of the study is to screen out an applicable and effective esophageal carcinoma preventive agent of traditional Chinese medicine, to offer academic foundation for further clinic study and to spread out its application.
Part 1 The observation of tumor-inhibitory effect of TCCP on the nude mice bearing esophageal carcinoma cells
Objective: To observe the inhibitory effect of TCCP on the nude mice bearing esophageal squmous carcinoma cells and explore the inhibitory mechanism in vivo.
Methods: Eca109-luc stablely expressing luciferase were inoculated subcutaneously into the hind flank region of nude mice to establish tumor model. Nude mice were randomly grouped until tuomr’s length reached 5-7 mm. One group of mice (n=7) was treated i.s. with NS. A second group of mice (n=7) was treated i.s with TCCP (20mg/kg) thrice per week. The course of all the treatments would last for 4 weeks. Tumor growth were monitored with in vivo imaging system. Cell apoptosis in vivo were analyzed by flow cytometry. Western blot was used to investigate protein expression of Survivin.
Results: 1 Compared with NS control group, the tumor growth of the nude mice bearing Eca109-luc was significantly inhibited by TCCP treatment (1.38±0.36 g vs 0.82±0.14 g), with the inhibitory ratio 40.7%.
2 There was no significant difference in photon number detected by in vivo bioluminescence imaging between NS control group and TCCP group before treatment with TCCP (5.02E+06±1.03E+06, 5.47E+06±1.12E+06 respectively)( P>0.05). After treatment with TCCP, the average photon number of the TCCP group were decreased by 7.65E+06±2.14E+06、6.18E+07±1.36E+07、1.70E+08±1.14+08 and 5.53E+08±1.01E+08 at weeks 1, 2, 3 and 4, respectively, significantly lower than NS control group (6.48E+07±1.25E+07、3.65E+08±1.42E+08、7.64E+08±3.02E+07 and 3.34E+9±4.78E+08, respectively) (P<0.01).
3 FCM assay for the apoptosis rate of tumor cells in vivo showed that there was a significant difference between the NS control group (11.6±1.0%) and the TCCP group (32.5±1.2%) (P<0.05).
4 Gene expression of Fas in TCCP treated mice was significantly higher than that of NS group(P<0.01), while survivin mRNA was significantly decreased.
5 It was observed that the protein expreesion of Fas in the tumor tissues of TCCP group was significantly higher (0.725±0.046), as compared with that of NS control group (0.297±0.026)(P<0.01), while the protein expreesion of Survivin was significantly decreased (0.052±0.003), as compared with that of NS control group (0.387±0.016)(P<0.05).
Conclusions: Cell growth in vivo was inhibited significantly by TCCP which might exert its anti-tumor activity through Fas/FasL pathway related to reduction of survivin expression, and then inducing apoptosis of tumor cells.
Part 2 Effect of TCCP on NMBA-induced rats esophageal carcinogenesis and its mechanism
1 The establish of rat model of esophageal preneoplastic lesion and the effect of TCCP on the cacinogenesis
Objective: To investigate the effect of TCCP on esophageal pathology , proliferating cell muclear antigen (PCNA) expression and cell cycle regulatory components in NMBA-induced rat tumorigenesis.
Methods: 165 male F344 rats (5-6 weeks of age) were randomly divided into five experimental groups according to the different regiments. It consisted of the following groups: (1) normal control groups (group 1, n=15): rats were raised rountinly; (2) soya oil control groups (group 2, n=15): rats were injected with soya oil 1ml/kg intramuscularily (i.m.); (3) NMBA control groups (group 3, n= 45): rats were injected with NMBA 0.5mg/kg only; (4) Low dose TCCP treatment groups (group 4, n=45): rats received NMBA 0.5mg/kg s.c. plus TCCP 20mg/kg i.m.; (5) High dose TCCP treatment groups (group 5, n=45): rats received NMBA 0.5mg/kg s.c. plus TCCP 10mg/kg i.m..The administration of drugs was scheduled as below: thrice per week for 5 weeks. At 9, 15 and 25 week, 5 rats from groups 1 and 2 and 15 rats from groups 3, 4 and 5 were euthanized by pentobarbital sodium and subjected to gross necropsy. The esophagus of each rat was excised and opened longitudinally. Then esophagus were fixed in 10% phosphate-buffer formalin solution, and routinely embedded in paraffin for HE staining to observe the pathological changes of esophageal tissues, while the expression of PCNA, cyclinD1 and CDK4 was measured by immunohistochemistry.
Results: 1 General observations. The mean body weights and food consumption in all rats were not significantly different throughout the bioassay. At the end of the bioassay rats treated with NMBA were found in low spirits with shaggy fur. However, body weights still increased.
2 General observations of esophagi of rats. There were no abnormal changes in normal and soya oil group among bioassay. Changes were observed in NMBA-induced rats as following: (1) esophageal mucosa becoming incrassation with color white and scabrities; (2) white patch. there were some pathologic white patch changes on the surface of esophageal mucosa just like dot, circle, orbicular-ovate or strip with size about 0.1-0.2 mm and the area of the pathologic changes increased at the end of bioassay. (3) papilloma. At week 9 of the bioassay, esophagi had pathologic changes like incrassation and white patch. At weeks 15 and 25 of the bioassay, 20% to 60% of the esophagi, respectively, had papillomas. Mean number of papillomas per rat is 0.93 at week 25.TCCP showed significant inhibitory effects on the incidence of the papilloma in group 4 to 20% ( P<0.01), and the mean number of papillomas was also decreased to 0.27. In group 5, the decrease of incidence and number of the papilloma was not statistically significant (33.3%, 0.33, respectively). There was also no statistically significant between group 4 and group 5.
3 The results of histopathologic examination of esophagi. The preneoplatic lesion of esophageal tissues was evaluated. At weeks 9, 15 and 25, the incidence of preneoplastic lesion in groups 3 was 20.0%, 46.7% and 93.3 %, respectively. TCCP markedly inhibited rat esophageal preneoplastic lesion, the inhibitory rates at 25 weeks were 64.3% in group 4 and 50% in group 5, respectively (P<0.05).
4 There were a markedly increase in the expression of PCNA in NMBA control group (week 9: 213.17±29.74; week 15: 268.35±39.56; week 25: 327.24±28.19), as compared with that in normal control group(167.96±20.16、170.76±14.79、172.49±17.49, respectively)( P <0.05). TCCP significantly decreased the expression of PCNA in groups 4 (185.28±22.98、200.56±28.19、282.23±37.43) and group 5 (189.27±22.10、209.73±28.88、305.60±17.46) (P<0.05).
5 The expreesion of CDK4 and cyclin D1 were not found in normal mucosa of esophagi of rats in normal control and soya oil control group, but were induced by administering NMBA. Positive expression of cyclinD1 and CDK4 increased gradually and reached the peak at 25wk. There were significant positive correlations between the two protein expression (r=0.9631, P<0.05). Treatment with TCCP could significantly decrease the protein expression of CDK4 at weeks 9, 15 and 25 (P<0.05). And there was significant difference between the high TCCP group and low TCCP group at weeks 15 and 25. TCCP could act a similar effect on the expreesion of cyclinD1.
Conclusions: NMBA (0.5mg/kg) can sucessfully induced F344 rats model of esophageal preneoplastic lesion. Typical histophathologic changes was examined in the NMBA-treated rats. TCCP may restrain the high expression of PCNA, while alleviate the esophageal preneoplastic lesion of rats induced by NMBA. TCCP can also inhibit the uncontrolled proliferation of esophageal epithelia cells and regulating cell-cycle process.
2 The effect of TCCP on wnt signal pathway in NMBA-induced rat tumorigenesis
Objective: To study the effect of TCCP on wnt signal pathway in NMBA-induced rats tumorigenesis and to investigate the mechanism by which TCCP modulate tumorigenesis.
Methods: Total cellular RNA was isolated from frozen tissues using TRIzol Reagent. The expression of c-myc mRNA was detected by RT-PCR. Western blot was used to investigate the protein expression of GSK-3βandβ-catenin in the esophageal epithelium.
Results: 1 The gene expression of c-myc of esophageal epithelium in NMBA control group was significantly increased at weeks 9, 15, 25 compared with normal control (P<0.05). TCCP supressed the mRNA expression of c-myc at both weeks 9 and 15 (P<0.05), but not at weeks 25.There was no significant difference between the high TCCP group and low TCCP group.
2 It was observed that the expression ofβ-catenin was slightly present in normal mucosa of esophagi of the rats which were untreated with NMBA, and then was significantly increased by administering NMBA from weeks 9 to 25 (P<0.05). The up-regulatedβ-catenin expression was decreased significantly by TCCP treatment at each check-point (P<0.05). The expression of GSK-3βwas rich in normal mucoa of esophagus, but was decreased significantly by administering NMBA, and was lowest at 25th week (P<0.05). The decreased protein levels of GSK3βwas significantly elevated by TCCP treatment at each check-point (P<0.05). But there was no significantly between the two treatment groups.
Conclusions: TCCP inhibited NMBA-induced rat esophageal carcinogenesis probably via acitivition of GSK-3β, suppression ofβ-catenin and c-myc expression.
Part 3 Effects of triterpenes compound of Cortex Periplocae on cyto-immunologic function in N-nitrosomethylbenzylamine-induced esophageal tumorigenesis in F344 rats
Objective: To investigate the mechanism and effects of triterpenes compound of Cortex Periplocae on cyto-immunologic function in N-nitrosomethyl- benzylamine-induced esophageal tumorigenesis in F344 rats.
Methods: 1 FCM and RT-PCR were used to detected the proportion of CD4+CD25+ and gene expression of Foxp3 in peripheral blood of rats . 2 RT-PCR and ELASA were used to detected the gene and protein expression of IL-2, IL-10 and TGF-β1 in peripheral blood of rats .
Results: 1 At week 9 after the NMBA treatment, CD4+T cells in peripheral blood of NMBA group rats were decreased significantly and CD4+CD25+ regulatory T cells and Foxp3 mRNA in peripheral blood of NMBA group rats were significantly increased, which deteriorated further with the time going on (P<0.05). At weeks 9 and 15, TCCP treatment can decreased the proportions of CD4+CD25+ regulatory T cells and the gene expression of Foxp3, while the proportions of CD4+ cells were significantly increased (P<0.05), compared with NMBA group. At week 25, TCCP significantly decreased the expression of Foxp3 mRNA, but had no significant effect on CD4+CD25+Treg and CD4+T cell.
At week 9 after NMBA treatment, the protein levels of TGF-β1 and IL-10 in peripheral blood of in F344 rats were significantly higher than those in normal control group and soy control group. The gene expression of TGF-β1 and IL-10 were also significantly elevated. While the levels of protein and gene expression of IL-2 were significantly decreased, as compared with normal control. TCCP can decrease the expression of IL-10 and TGF-β1, increase the expression of IL-2.
Conclusions: The expression of CD4+CD25+ regulatory T cells and Foxp3 mRNA in peripheral blood of rats were enhanced after treatment with NMBA, while the triterpenes compound of Cortex Periplocae can effectively inhibit the expression, thus it obviously improve the immune dysfunction in experimental esophageal tumorigenesis in F344 rats.
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