消痰散结方对裸鼠人胃癌模型淋巴管生长因子影响的实验研究
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摘要
背景
淋巴转移是胃癌转移的主要途径之一,也是影响术后复发及患者生存率的重要因素,有研究发现,在发生淋巴结转移的肿瘤中,常有淋巴管的增生和扩张,表明淋巴管生成在肿瘤淋巴转移过程中起着不容忽视的作用。
导师魏品康教授积数十年临床经验,在总结前人理论基础上,构建胃癌“痰证学说”,认为“痰”是肿瘤发生、发展、转移的关键物质基础,因此提出“消痰散结”作为胃癌的核心治则,创立“消痰散结方”并应用于临床,取得了明显疗效。
我科前期研究发现消痰散结方通过多靶点,多途径抑制肿瘤增殖,预防肿瘤复发、转移,但该方对于胃癌淋巴转移这一重要转移途径研究尚未深入,故本课题在前期研究的基础上,通过复制裸鼠人胃癌MKN-45原位移植模型,观察消痰散结方对胃癌淋巴管生长因子表达的影响,进一步探讨消痰散结方在防治胃癌淋巴转移方面的作用机理。
目的
通过复制建立裸鼠人胃癌MKN-45原位移植瘤模型,观察肿瘤生长、转移情况,计算抑瘤率,并通过运用EnVision、RT-PCR等方法,检测消痰散结方对肿瘤组织中VEGF-C,VEGF-D和VEGFR-3蛋白和mRNA表达的调控作用,探讨消痰散结方在防治肿瘤淋巴转移方面的作用机制。
方法
1.选取经传代后生长良好的人胃癌MKN-45细胞瘤株,将其移植到BALB／C裸小鼠胃大弯近胃窦旁,建立裸鼠人胃癌移植瘤模型。定期观察裸鼠全身情况和腹部瘤块生长特征,待动物出现衰竭征象时处死,解剖观察移植瘤局部生长情况。
2.40只BALB／C雄性裸小鼠,采用随机数字表法分为：生理盐水组、5-Fu组、榄香烯组和消痰散结方组,术后48h后给予不同的干预：分别给予生理盐水0.4ml灌胃,1／日；5-Fu稀释液0.2ml腹腔注射(60mg.kg-1.w-1),1／周×3周：榄香烯稀释液0.4ml (2.5mg/ml)灌胃,1／日；消痰散结方0.4ml灌胃,1／日；给药时间为期6周,定期观察裸鼠全身情况、体重变化和腹部体征。
3.第6周实验结束时,麻醉后牺牲、解剖实验动物,观察移植瘤局部生长情况及周围组织转移情况,剥取瘤组织,称取瘤重,计算抑瘤率。行HE染色,对肿瘤进行病理诊断。
4.取瘤组织,行EnVision免疫组化方法,半定量分析和比较肿瘤组织VEGF-C,VEGF-D和VEGFR-3蛋白的表达,计数LMVD,分析肿瘤微淋巴管生成的情况。
5.取瘤组织抽提总mRNA,逆转录合成cDNA行RT-PCR检测,从mRNA水平检测和比较VEGF-C,VEGF-D和VEGFR-3 mRNA的表达变化。
结果
1.成功建立裸鼠人胃癌MKN-45原位移植瘤模型。移植术后10天左右左上腹部可扪及直径约为5mm左右质地较硬的结节,随后结节逐渐增大,6周时部分荷瘤鼠出现消瘦、行动迟缓等衰竭症状,原位移植瘤突起于腹壁,质硬,部分动物瘤体突出至皮下,瘤块透壁可见,解剖后可见胃壁上有灰白色鱼肉状的肿瘤组织,呈实质性,膨胀性生长,有包膜,表面多呈椭圆形结节状,高低不平,质地较硬,粘膜下有血管走行,胃被包绕在瘤块中。部分原位瘤与肝、脾及腹壁粘连。裸鼠移植瘤原位成瘤率100%,HE染色证实为低分化腺癌。
2.各组动物均可观察到腹腔淋巴结肿大,其中5-Fu组,榄香烯组,消痰散结方组出现淋巴结转移比率分别为60%,40%,40%,明显低于生理盐水组90%。
3.消痰散结方对MKN-45荷瘤小鼠肿瘤生长抑制情况表明：5-Fu组,榄香烯组,消痰散结方组的抑瘤率分别为66.5%、51.0%,57.1%。5-Fu组,榄香烯组,消痰散结方组的平均瘤重明显小于生理盐水组(P<0.01)；消痰散结方组平均瘤重与5-Fu组,榄香烯组相比差异无统计学意义(P>0.05),5-Fu组平均瘤重与榄香烯组相比差异有统计学意义(P<0.05)。
4.消痰散结方对移植瘤组织中VEGF-C蛋白及mRNA表达的影响：
(1)消痰散结方对移植瘤组织中VEGF-C蛋白表达的影响：与生理盐水组相比,消痰散结方组、5-Fu组、榄香烯组均可明显降低VEGF-C的阳性表达(P<0.01),三组药物之间互相比较：消痰散结方组与榄香烯组相比,差异有统计学意义(P<0.05)；消痰散结方组、榄香烯组与5-Fu组相比,差异无统计学意义(P>0.05)。
(2)消痰散结方对移植瘤组织中VEGF-C mRNA表达的影响：在VEGF-C mRNA的表达上消痰散结方组、5-Fu组、榄香烯组的cDNA扩增条带面积灰度值均较生理盐水组明显下调,(P<0.05),三组药物之间互相比较差异均无统计学意义(P>0.05)。
5.消痰散结方对移植瘤组织中VEGF-D蛋白及mRNA表达的影响：
(1)消痰散结方对移植瘤组织中VEGF-D蛋白表达的影响：与生理盐水组相比,消痰散结方组、5-Fu组、榄香烯组均可明显降低VEGF-D的阳性表达(P<0.01),三组药物之间互相比较差异均无统计学意义(P>0.05)。
(2)消痰散结方对移植瘤组织中VEGF-D mRNA表达的影响：在VEGF-D mRNA的表达上消痰散结方组、5-Fu组、榄香烯组的cDNA扩增条带面积灰度值均较生理盐水组明显下调,(P<0.05),三组药物之间互相比较差异均无统计学意义(P>0.05)。
6.消痰散结方对移植瘤组织中VEGFR-3蛋白及mRNA表达的影响：
(1)消痰散结方对移植瘤组织中VEGFR-3蛋白表达的影响：与生理盐水组相比,消痰散结方组、5-Fu组、榄香烯组均可降低VEGF-3的阳性表达(P<0.05),消痰散结方组与5-Fu组、榄香烯组相比,差异均无统计学意义(P>0.05)。
(2)消痰散结方对移植瘤组织中VEGFR-3 mRNA表达的影响：在VEGFR-3 mRNA的表达上消痰散结方组、5-Fu组、榄香烯组的cDNA扩增条带面积灰度值均较生理盐水组明显下调(P<0.05),三组药物之间互相比较差异均无统计学意义(P>0.05)
7.消痰散结方对移植瘤组织中微淋巴管密度的影响：各组LMVD值计数分别为生理盐水组17.43±1.79,5-Fu组13.33±2.71,榄香烯组10.20±3.64,消痰散结方组12.23±3.21,与生理盐水组相比较,消痰散结方组、5-Fu组、榄香烯组均可明显降低移植瘤组织中的微淋巴管密度(P<0.05),三组药物之间互相比较：消痰散结方组与5-Fu组、榄香烯组相比差异均无统计学意义(P>0.05)
结论：
1.消痰散结方对裸鼠人胃癌MKN-45原位移植瘤增殖和转移具有明显抑制作用；
2.消痰散结方可明显降低移植瘤组织中微淋巴管密度,可能是其抑制肿瘤淋巴转移的作用途径；
3.消痰散结方能够不同程度地下调淋巴管生长因子VEGF-C、VEGF-D及其受体在蛋白水平和mRNA水平的表达,可能是其抑制肿瘤淋巴转移的分子生物学机制。
Background:
Lymphatic System is a major route of metastasis in gastric carcinoma, and lymph node metastasis is also one of the major prognostic factors. Studies have found that in the event of lymph node metastasis of tumors, lymphatic proliferation and expansion often can be observed.This indicates that lymphangiogenesis plays an important role in tumor metastasis.
In his long-term medical practice, Professor Wei-Pinkang has established his theory of "Phlegm theory" by integrating modern medicine and Traditional Chinese Medicine treatment of gastric carcinoma, he consider that "Phlegm" is material foundation of generation and metastasis in tumor. He believes that "dispersing phlegm" is a basic treatment for gastric carcinoma, and creates Xiaotan Sanjie Recipe, which is applied to gastric carcinoma treatment and achieves satisfactory results. In prior studies, we have found that Xiaotan Sanjie Recipe can inhibit tumor growth and metastasis.But the mechanism of inhibiting lymph node metastasis in gastric carcinoma is not clear,so the purpose of this study is to discover the mechanism of Xiaotan Sanjie Recipe in inhibiting gastric carcinoma lymphatic metastasis on gastric carcinoma orthotropic transplantation model.
Objective:
Through the establishment of MKN-45 human gastric carcinoma orthotopic transplantation tumor model in nude mice,to discover the mechanism of Xiaotan Sanjie Recipe in inhibiting gastric carcinoma lymphatic metastasis by observing the expression of VEGF-C,VEGF-D and VEGFR-3 on gastric carcinoma orthotropic implantation model.All the factors were detected by immunohistochemical staining, RT-PCR, and other molecular biology methods.
Methods:
1. The MKN-45 cancer cell was orthotopic implanted on stomach near the greater curvature side in BALB/C nude mice,nude mice transplantation tumor model of human gastric carcinoma was set up, the growth of tumor and the life status of mice were observed regularly. The animals were sacrificed and the local growth of transplanted tumor was observed.
2.40 mice were divided into 4 groups:saline group,5-Fu group, Elemene group, and Xiaotan Sanjie Recipe group randomly after the orthotopic transplantation model was built. The administrations began from the 48h after the surgery, which were as follows: the mice in saline group were given isotonic saline 0.4ml ig. once a day×6w; those in 5-Fu group were given 5-Fu dilution(60mg.kg-1.w-1) 0.2ml ip. once a week×3w; those in Elemene group were given Elemene 0.4ml (2.5mg/ml) ig. once a day×6w; and those in Xiaotan Sanjie Recipe group were given Xiaotan Sanjie Recipe 0.4ml ig. once a day×6w. The life status, weight change and abdominal signs were observed regularly
3.The experiment was terminated on the 6th week. The mice were anesthetized, sacrificed and dissected, the tumor metastasis to the surrounding tissue was measured, the tumors were weighted, observed pathologic diagnosis,inhibition rate was calculated.
4. The expressions of VEGF-C,VEGF-D and VEGFR-3 were detected and LMVD was counted and analyzed semi-quantitatively by means of Envision immunohistochemical staining in tumor.
5. The mRNA was extracted from tumors for the cDNA synthesizing. Detect the mRNA expression of VEGF-C,VEGF-D and VEGFR-3 by means of RT-PCR.
Results:
1. We built an orthotopic implantation model of human gastric carcinoma MKN-45 in nude mice successfully.10 days after surgery, the lump could be touched in the upper abdominal about 5mm. On the 6rd week, the lump appeared and looked noduous through the skins. Gradually nude appeared deprivation and weight loss, slow action, such as failure symptoms. The abdomen opened, the tumors were gray-white and fish-shaped tumor tissues were the substantive, which were round or oval. The stomachs of mice were surrounded by the tumor tissues, and the some tissues adhered to the adjacent organs such as liver, gut, lungs and lymphaden. The formation rate was 100%. The HE staining illustrated the tumors was low-differentiated adencarcinoma.
2. Animals in each group could be observed in enlarged abdominal lymph nodes, lymph node metastasis rates of 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group were 60%,40%,40%, significantly lower than saline group,which was 90%.
3. The tumor inhibition rate was 66.5%in 5-Fu group,51.0%in Elemene group and 53.25%in Xiaotan Sanjie Recipe group. Compared with saline group, average tumor weight of 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was lower significantly (P< 0.01); Compared with Xiaotan Sanjie Recipe group,5-Fu group and Elemene group had no significant difference(P>0.05); the difference between 5-Fu group and Elemene group was significant (P<0.05);
4. The results of Xiaotan Sanjie Recipe affecting VEGF-C on nude mice tumor tissue:
(1) The results of Xiaotan Sanjie Recipe affecting the expression of VEGF-C protein showed:Compared with saline group, the VEGF-C expression in 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.01); the difference between Xiaotan Sanjie Recipe group and Elemene group had a statistical significance (P<0.05); Compared with 5-Fu group, Xiaotan Sanjie Recipe group and Elemene group had no significant difference (P> 0.05)
(2) The results of Xiaotan Sanjie Recipe affecting the expression of endostatin mRNA showed:Compared with saline group, the VEGF-C mRNA expression in 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.05); 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group compared with each other had no statistical significance (P >0.05).
5. The results of Xiaotan Sanjie Recipe affecting VEGF-D on nude mice tumor tissue:
(1) The results of Xiaotan Sanjie Recipe affecting the expression of VEGF-D protein showed:Compared with saline group, the VEGF-D expression in 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.01); 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group compared with each other had no statistical significance (P>0.05).
(2) The results of Xiaotan Sanjie Recipe affecting the expression of endostatin mRNA showed:Compared with saline group, the VEGF-D mRNA expression in 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.05); 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group compared with each other had no statistical significance (P >0.05).
6. The results of Xiaotan Sanjie Recipe affecting VEGFR-3 on nude mice tumor tissue:
(1) The results of Xiaotan Sanjie Recipe affecting the expression of VEGFR-3 protein showed:Compared with saline group, the VEGFR-3 expression in 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.05); Compared with Xiaotan Sanjie Recipe group,5-Fu group and Elemene group had no significant difference (P>0.05);
(2) The results of Xiaotan Sanjie Recipe affecting the expression of endostatin mRNA showed:Compared with saline group, the VEGFR-3 mRNA expression in 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.05); 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group compared with each other had no statistical significance (P >0.05).
7. The results of Xiaotan Sanjie Recipe affecting lymphatic microvessel density:
LMVD count was as follows:saline group was 17.43±1.79,5-Fu group was 13.33±2.71, Elemene group was 10.20±3.64, and Xiaotan Sanjie Recipe group was 12.23±3.21. Compared with saline group, LMVD of 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.05); Compared with Xiaotan Sanjie Recipe group,5-Fu group and Elemene group had no significant difference (P>0.05);
Conclusion: 1. Xiaotan Sanjie Recipe can obviously inhibite tumor growth and lymphatic metastasis of the orthotopic transplantation model of human gastric carcinoma MKN-45 in nude mice.
2. Xiaotan Sanjie Recipe can decrease lymphatic microvessel density in gastric carcinoma, this may be the pathways that Xiaotan Sanjie Recipe inhibites lymph node metastasis of gastric carcinoma.
3. Xiaotan Sanjie Recipe can down-regulate the expressions of VEGF-C,VEGF-D and VEGFR-3 proteins and mRNAs,which may be one of the mechanisms inhibiting lymphatic metastasis.
淋巴转移是胃癌转移的主要途径之一,也是影响术后复发及患者生存率的重要因素,有研究发现,在发生淋巴结转移的肿瘤中,常有淋巴管的增生和扩张,表明淋巴管生成在肿瘤淋巴转移过程中起着不容忽视的作用。
导师魏品康教授积数十年临床经验,在总结前人理论基础上,构建胃癌“痰证学说”,认为“痰”是肿瘤发生、发展、转移的关键物质基础,因此提出“消痰散结”作为胃癌的核心治则,创立“消痰散结方”并应用于临床,取得了明显疗效。
我科前期研究发现消痰散结方通过多靶点,多途径抑制肿瘤增殖,预防肿瘤复发、转移,但该方对于胃癌淋巴转移这一重要转移途径研究尚未深入,故本课题在前期研究的基础上,通过复制裸鼠人胃癌MKN-45原位移植模型,观察消痰散结方对胃癌淋巴管生长因子表达的影响,进一步探讨消痰散结方在防治胃癌淋巴转移方面的作用机理。
目的
通过复制建立裸鼠人胃癌MKN-45原位移植瘤模型,观察肿瘤生长、转移情况,计算抑瘤率,并通过运用EnVision、RT-PCR等方法,检测消痰散结方对肿瘤组织中VEGF-C,VEGF-D和VEGFR-3蛋白和mRNA表达的调控作用,探讨消痰散结方在防治肿瘤淋巴转移方面的作用机制。
方法
1.选取经传代后生长良好的人胃癌MKN-45细胞瘤株,将其移植到BALB／C裸小鼠胃大弯近胃窦旁,建立裸鼠人胃癌移植瘤模型。定期观察裸鼠全身情况和腹部瘤块生长特征,待动物出现衰竭征象时处死,解剖观察移植瘤局部生长情况。
2.40只BALB／C雄性裸小鼠,采用随机数字表法分为：生理盐水组、5-Fu组、榄香烯组和消痰散结方组,术后48h后给予不同的干预：分别给予生理盐水0.4ml灌胃,1／日；5-Fu稀释液0.2ml腹腔注射(60mg.kg-1.w-1),1／周×3周：榄香烯稀释液0.4ml (2.5mg/ml)灌胃,1／日；消痰散结方0.4ml灌胃,1／日；给药时间为期6周,定期观察裸鼠全身情况、体重变化和腹部体征。
3.第6周实验结束时,麻醉后牺牲、解剖实验动物,观察移植瘤局部生长情况及周围组织转移情况,剥取瘤组织,称取瘤重,计算抑瘤率。行HE染色,对肿瘤进行病理诊断。
4.取瘤组织,行EnVision免疫组化方法,半定量分析和比较肿瘤组织VEGF-C,VEGF-D和VEGFR-3蛋白的表达,计数LMVD,分析肿瘤微淋巴管生成的情况。
5.取瘤组织抽提总mRNA,逆转录合成cDNA行RT-PCR检测,从mRNA水平检测和比较VEGF-C,VEGF-D和VEGFR-3 mRNA的表达变化。
结果
1.成功建立裸鼠人胃癌MKN-45原位移植瘤模型。移植术后10天左右左上腹部可扪及直径约为5mm左右质地较硬的结节,随后结节逐渐增大,6周时部分荷瘤鼠出现消瘦、行动迟缓等衰竭症状,原位移植瘤突起于腹壁,质硬,部分动物瘤体突出至皮下,瘤块透壁可见,解剖后可见胃壁上有灰白色鱼肉状的肿瘤组织,呈实质性,膨胀性生长,有包膜,表面多呈椭圆形结节状,高低不平,质地较硬,粘膜下有血管走行,胃被包绕在瘤块中。部分原位瘤与肝、脾及腹壁粘连。裸鼠移植瘤原位成瘤率100%,HE染色证实为低分化腺癌。
2.各组动物均可观察到腹腔淋巴结肿大,其中5-Fu组,榄香烯组,消痰散结方组出现淋巴结转移比率分别为60%,40%,40%,明显低于生理盐水组90%。
3.消痰散结方对MKN-45荷瘤小鼠肿瘤生长抑制情况表明：5-Fu组,榄香烯组,消痰散结方组的抑瘤率分别为66.5%、51.0%,57.1%。5-Fu组,榄香烯组,消痰散结方组的平均瘤重明显小于生理盐水组(P<0.01)；消痰散结方组平均瘤重与5-Fu组,榄香烯组相比差异无统计学意义(P>0.05),5-Fu组平均瘤重与榄香烯组相比差异有统计学意义(P<0.05)。
4.消痰散结方对移植瘤组织中VEGF-C蛋白及mRNA表达的影响：
(1)消痰散结方对移植瘤组织中VEGF-C蛋白表达的影响：与生理盐水组相比,消痰散结方组、5-Fu组、榄香烯组均可明显降低VEGF-C的阳性表达(P<0.01),三组药物之间互相比较：消痰散结方组与榄香烯组相比,差异有统计学意义(P<0.05)；消痰散结方组、榄香烯组与5-Fu组相比,差异无统计学意义(P>0.05)。
(2)消痰散结方对移植瘤组织中VEGF-C mRNA表达的影响：在VEGF-C mRNA的表达上消痰散结方组、5-Fu组、榄香烯组的cDNA扩增条带面积灰度值均较生理盐水组明显下调,(P<0.05),三组药物之间互相比较差异均无统计学意义(P>0.05)。
5.消痰散结方对移植瘤组织中VEGF-D蛋白及mRNA表达的影响：
(1)消痰散结方对移植瘤组织中VEGF-D蛋白表达的影响：与生理盐水组相比,消痰散结方组、5-Fu组、榄香烯组均可明显降低VEGF-D的阳性表达(P<0.01),三组药物之间互相比较差异均无统计学意义(P>0.05)。
(2)消痰散结方对移植瘤组织中VEGF-D mRNA表达的影响：在VEGF-D mRNA的表达上消痰散结方组、5-Fu组、榄香烯组的cDNA扩增条带面积灰度值均较生理盐水组明显下调,(P<0.05),三组药物之间互相比较差异均无统计学意义(P>0.05)。
6.消痰散结方对移植瘤组织中VEGFR-3蛋白及mRNA表达的影响：
(1)消痰散结方对移植瘤组织中VEGFR-3蛋白表达的影响：与生理盐水组相比,消痰散结方组、5-Fu组、榄香烯组均可降低VEGF-3的阳性表达(P<0.05),消痰散结方组与5-Fu组、榄香烯组相比,差异均无统计学意义(P>0.05)。
(2)消痰散结方对移植瘤组织中VEGFR-3 mRNA表达的影响：在VEGFR-3 mRNA的表达上消痰散结方组、5-Fu组、榄香烯组的cDNA扩增条带面积灰度值均较生理盐水组明显下调(P<0.05),三组药物之间互相比较差异均无统计学意义(P>0.05)
7.消痰散结方对移植瘤组织中微淋巴管密度的影响：各组LMVD值计数分别为生理盐水组17.43±1.79,5-Fu组13.33±2.71,榄香烯组10.20±3.64,消痰散结方组12.23±3.21,与生理盐水组相比较,消痰散结方组、5-Fu组、榄香烯组均可明显降低移植瘤组织中的微淋巴管密度(P<0.05),三组药物之间互相比较：消痰散结方组与5-Fu组、榄香烯组相比差异均无统计学意义(P>0.05)
结论：
1.消痰散结方对裸鼠人胃癌MKN-45原位移植瘤增殖和转移具有明显抑制作用；
2.消痰散结方可明显降低移植瘤组织中微淋巴管密度,可能是其抑制肿瘤淋巴转移的作用途径；
3.消痰散结方能够不同程度地下调淋巴管生长因子VEGF-C、VEGF-D及其受体在蛋白水平和mRNA水平的表达,可能是其抑制肿瘤淋巴转移的分子生物学机制。
Background:
Lymphatic System is a major route of metastasis in gastric carcinoma, and lymph node metastasis is also one of the major prognostic factors. Studies have found that in the event of lymph node metastasis of tumors, lymphatic proliferation and expansion often can be observed.This indicates that lymphangiogenesis plays an important role in tumor metastasis.
In his long-term medical practice, Professor Wei-Pinkang has established his theory of "Phlegm theory" by integrating modern medicine and Traditional Chinese Medicine treatment of gastric carcinoma, he consider that "Phlegm" is material foundation of generation and metastasis in tumor. He believes that "dispersing phlegm" is a basic treatment for gastric carcinoma, and creates Xiaotan Sanjie Recipe, which is applied to gastric carcinoma treatment and achieves satisfactory results. In prior studies, we have found that Xiaotan Sanjie Recipe can inhibit tumor growth and metastasis.But the mechanism of inhibiting lymph node metastasis in gastric carcinoma is not clear,so the purpose of this study is to discover the mechanism of Xiaotan Sanjie Recipe in inhibiting gastric carcinoma lymphatic metastasis on gastric carcinoma orthotropic transplantation model.
Objective:
Through the establishment of MKN-45 human gastric carcinoma orthotopic transplantation tumor model in nude mice,to discover the mechanism of Xiaotan Sanjie Recipe in inhibiting gastric carcinoma lymphatic metastasis by observing the expression of VEGF-C,VEGF-D and VEGFR-3 on gastric carcinoma orthotropic implantation model.All the factors were detected by immunohistochemical staining, RT-PCR, and other molecular biology methods.
Methods:
1. The MKN-45 cancer cell was orthotopic implanted on stomach near the greater curvature side in BALB/C nude mice,nude mice transplantation tumor model of human gastric carcinoma was set up, the growth of tumor and the life status of mice were observed regularly. The animals were sacrificed and the local growth of transplanted tumor was observed.
2.40 mice were divided into 4 groups:saline group,5-Fu group, Elemene group, and Xiaotan Sanjie Recipe group randomly after the orthotopic transplantation model was built. The administrations began from the 48h after the surgery, which were as follows: the mice in saline group were given isotonic saline 0.4ml ig. once a day×6w; those in 5-Fu group were given 5-Fu dilution(60mg.kg-1.w-1) 0.2ml ip. once a week×3w; those in Elemene group were given Elemene 0.4ml (2.5mg/ml) ig. once a day×6w; and those in Xiaotan Sanjie Recipe group were given Xiaotan Sanjie Recipe 0.4ml ig. once a day×6w. The life status, weight change and abdominal signs were observed regularly
3.The experiment was terminated on the 6th week. The mice were anesthetized, sacrificed and dissected, the tumor metastasis to the surrounding tissue was measured, the tumors were weighted, observed pathologic diagnosis,inhibition rate was calculated.
4. The expressions of VEGF-C,VEGF-D and VEGFR-3 were detected and LMVD was counted and analyzed semi-quantitatively by means of Envision immunohistochemical staining in tumor.
5. The mRNA was extracted from tumors for the cDNA synthesizing. Detect the mRNA expression of VEGF-C,VEGF-D and VEGFR-3 by means of RT-PCR.
Results:
1. We built an orthotopic implantation model of human gastric carcinoma MKN-45 in nude mice successfully.10 days after surgery, the lump could be touched in the upper abdominal about 5mm. On the 6rd week, the lump appeared and looked noduous through the skins. Gradually nude appeared deprivation and weight loss, slow action, such as failure symptoms. The abdomen opened, the tumors were gray-white and fish-shaped tumor tissues were the substantive, which were round or oval. The stomachs of mice were surrounded by the tumor tissues, and the some tissues adhered to the adjacent organs such as liver, gut, lungs and lymphaden. The formation rate was 100%. The HE staining illustrated the tumors was low-differentiated adencarcinoma.
2. Animals in each group could be observed in enlarged abdominal lymph nodes, lymph node metastasis rates of 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group were 60%,40%,40%, significantly lower than saline group,which was 90%.
3. The tumor inhibition rate was 66.5%in 5-Fu group,51.0%in Elemene group and 53.25%in Xiaotan Sanjie Recipe group. Compared with saline group, average tumor weight of 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was lower significantly (P< 0.01); Compared with Xiaotan Sanjie Recipe group,5-Fu group and Elemene group had no significant difference(P>0.05); the difference between 5-Fu group and Elemene group was significant (P<0.05);
4. The results of Xiaotan Sanjie Recipe affecting VEGF-C on nude mice tumor tissue:
(1) The results of Xiaotan Sanjie Recipe affecting the expression of VEGF-C protein showed:Compared with saline group, the VEGF-C expression in 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.01); the difference between Xiaotan Sanjie Recipe group and Elemene group had a statistical significance (P<0.05); Compared with 5-Fu group, Xiaotan Sanjie Recipe group and Elemene group had no significant difference (P> 0.05)
(2) The results of Xiaotan Sanjie Recipe affecting the expression of endostatin mRNA showed:Compared with saline group, the VEGF-C mRNA expression in 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.05); 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group compared with each other had no statistical significance (P >0.05).
5. The results of Xiaotan Sanjie Recipe affecting VEGF-D on nude mice tumor tissue:
(1) The results of Xiaotan Sanjie Recipe affecting the expression of VEGF-D protein showed:Compared with saline group, the VEGF-D expression in 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.01); 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group compared with each other had no statistical significance (P>0.05).
(2) The results of Xiaotan Sanjie Recipe affecting the expression of endostatin mRNA showed:Compared with saline group, the VEGF-D mRNA expression in 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.05); 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group compared with each other had no statistical significance (P >0.05).
6. The results of Xiaotan Sanjie Recipe affecting VEGFR-3 on nude mice tumor tissue:
(1) The results of Xiaotan Sanjie Recipe affecting the expression of VEGFR-3 protein showed:Compared with saline group, the VEGFR-3 expression in 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.05); Compared with Xiaotan Sanjie Recipe group,5-Fu group and Elemene group had no significant difference (P>0.05);
(2) The results of Xiaotan Sanjie Recipe affecting the expression of endostatin mRNA showed:Compared with saline group, the VEGFR-3 mRNA expression in 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.05); 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group compared with each other had no statistical significance (P >0.05).
7. The results of Xiaotan Sanjie Recipe affecting lymphatic microvessel density:
LMVD count was as follows:saline group was 17.43±1.79,5-Fu group was 13.33±2.71, Elemene group was 10.20±3.64, and Xiaotan Sanjie Recipe group was 12.23±3.21. Compared with saline group, LMVD of 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.05); Compared with Xiaotan Sanjie Recipe group,5-Fu group and Elemene group had no significant difference (P>0.05);
Conclusion: 1. Xiaotan Sanjie Recipe can obviously inhibite tumor growth and lymphatic metastasis of the orthotopic transplantation model of human gastric carcinoma MKN-45 in nude mice.
2. Xiaotan Sanjie Recipe can decrease lymphatic microvessel density in gastric carcinoma, this may be the pathways that Xiaotan Sanjie Recipe inhibites lymph node metastasis of gastric carcinoma.
3. Xiaotan Sanjie Recipe can down-regulate the expressions of VEGF-C,VEGF-D and VEGFR-3 proteins and mRNAs,which may be one of the mechanisms inhibiting lymphatic metastasis.
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1.汤钊猷.现代肿瘤学[M].第2版.上海：上海医科大学出版社,2000,710.
2. Yonemura Y, Fushida S, Bando E, et al. Lymphangiogenesis and the vascular endothelial growth factor receptor (VEGFR)-3 in gastric cancer[J]. Eur J Cancer.2001 May;37(7):918-23.
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