启膈方抗胃癌转移机制的研究
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摘要
胃癌是常见的恶性肿瘤,是消化系发病率最高的肿瘤,约占消化道肿瘤的62%。我国是胃癌高发国家,发病率远高于欧美西方国家。我国每年约有17万人死于胃癌,占消化系统肿瘤死亡人数50%以上。尽管随着医疗水平的提高,综合治疗的运用,胃癌的5年生存率有所提高,但总的来说疗效仍不尽人意。究其原因胃癌早期无典型表现,患者就诊时多为进展期胃癌,手术治疗后多出现转移而导致治疗失败。据报道约40%—60%的患者发生胃癌复发和转移,寻求防治胃癌转移的可靠方法和药物是我们面对的迫切问题。
胃癌转移的主要途径为血道转移、淋巴道转移、腹膜种植转移。其中血道转移的危害最大,通过血液循环系统胃癌细胞可在其他脏器形成转移灶,破坏脏器功能最终导致死亡。肝、肺是胃癌血道转移最易发生的器官,文献报道胃癌肝转移的发生率为40-50%,肺转移的发生率为40%左右。肝、肺转移后多以全身化疗为主,尽管结合中药、手术、放疗、免疫治疗,其中位生存时间也仅为6-9月。由此可见,防止转移的发生是治疗胃癌的重要课题,对延长胃癌生存期有重要意义。
肿瘤转移的机制及抗肿瘤转移药物的研究是肿瘤研究的热点,肿瘤转移的基本过程已大概清楚,抗肿瘤转移药物集中在抗新生血管、抗血小板聚集、抗血小板活化、抑制转移基因表达、提高免疫等方面,多数处于实验研究阶段,而且这些药物作用途径、作用靶点单一,对于多步骤、多环节、多因素的胃癌转移过程,其抑制转移的作用显得杯水车薪。中药复方是在辩证论治的基础上的合理组合,它含有多种复杂成分,有多种治疗作用。由此,我们从转移的基本过程及相关因素,研究中药复方启膈方的抗转移作用。
启膈方是根据《医学心悟》中治疗噎嗝的启膈散化裁而成,临床用于治疗胃癌、食管癌患者。发现启膈方用于晚期胃癌患者可延长生存时间,防止、延缓转移灶的出现;用于胃癌术后患者可防止转移,延长无病生存时间。于是我们考虑该方有抗胃癌转移的作用,本研究从胃癌血道转移的过程入手,探讨启膈方抑制转移的作用环节及作用靶点。
本课题从体外实验、体内实验两个方面研究启膈方的抗转移作用。体外培养高侵袭胃癌、食管癌瘤株,启膈方干预后,观察对肿瘤细胞转移能力的影响。体内动物实验,用高肺转移小鼠胃癌瘤株FC接种615小鼠后灌服启膈方,观察肺转移抑制率及对相关转移因子的影响。
本课题分为四部分完成:
第一部分启膈方对人胃癌细胞MGC、人食管癌细胞EC109黏附的影响
目的:观察启膈方对人胃癌细胞株MGC与人胃癌细胞株MGC之间、人食管癌细胞株EC109与人食管癌细胞株EC109之间同质黏附的影响。用不同方法检测比较启膈方组、5FU组、空白对照组的黏附率、聚集体形成率及E-钙黏蛋白表达的差别。
方法:复苏人胃癌细胞株MGC、人食管癌细胞株EC109,待细胞呈对数生长期时,随机分为5组。启膈组加入启膈方,使其终浓度为15mg/ml、30mg/ml、60mg/ml,定为启膈方小、中、大剂量组。5FU组加入5FU,使其终浓度为50ug/ml,对照组加入PBS。用机械法检测各组细胞20、40、60分钟黏附情况,计算黏附率。用3-H-Tdr标记的放射性核素法检测各组细胞30、60分钟时液闪仪测定的放射强度,计算黏附率。用机械法检测各组细胞2、4、6、12小时聚集体形成情况,计算聚集体形成率,并测量各组细胞聚集体的最大面积。流式细胞术测定各组E-钙黏连蛋白的相对表达量。
结果:机械法检测MGC胃癌细胞,EC109食管癌细胞在20min、40min、60min的黏附率。启膈方各剂量组的黏附率均高于5FU组、空白对照组。启膈方各剂量组与空白对照组、5FU组之间差异有显著性(P<0.01),空白对照组、5FU组之间差异无显著性(P>0.05),启膈方各剂量组之间差异无显著性(P>0.05)。放射核素法检测MGC胃癌细胞、EC109食管癌细胞30分钟、60分钟黏附率,启膈方各剂量组均高于5FU组、空白对照组,启膈方各剂量组与空白对照组、5Fu组之间差异有显著性(P<0.05),空白对照组、5FU组之间差异无显著性(P>0.05),启膈方各剂量组之间差异无显著性(P>0.05)。MGC细胞、EC109细胞启膈方各剂量组的各时间点聚集体形成率、聚集体面积均大于空白对照组、5FU组(P<0.05),5FU组小于空白对照组(P<0.05),启膈方各剂量组之间差异无显著性(P>0.05)。流式细胞仪检测E-钙黏蛋白含量启膈方各剂量组均高于空白对照组、5FU组(P<0.05),空白对照组与5FU组之间差异无显著性(P>0.05),启膈方各剂量组之间差异无显著性(P>0.05)。
结论:启膈方可提高MGC人胃癌细胞、EC109人食管癌细胞的黏附率、聚集体形成率、提高了同质黏附能力,5FU对同质黏附率无明显影响,长时间作用于肿瘤细胞后影响聚集体的形成。启膈方可提高MGC人胃癌细胞、EC109人食管癌细胞E-Cadherin蛋白的表达。
第二部分启膈方对人胃癌细胞MGC、人食管癌细胞EC109增殖、运动、分泌MMP-2、MMP-9的影响
目的:观察启膈方对MGC、EC109细胞增殖能力、运动能力、分泌MMP-2、MMP-9的抑制作用,比较5FU组MGC、EC109细胞增殖抑制率,运动速度、MMP-2、MMP-9活性的不同。
方法:MTT法检测肿瘤细胞的增殖。在96孔板上每孔加入细胞悬液100ul,细胞数为2×103,分为启膈方、5FU和空白对照组,启膈方组分别加入启膈方,使其终浓度为15mg/ml、30mg/ml、60mg/ml定为小、中、大剂量组,5FU组加入5FU注射液,使其终浓度为50ug/ml,在空白对照组加入PBS。培养箱中孵育24、48、72h,培养结束前4小时加入MTT,检测各时间点的OD值,计算增殖抑制率。琼脂滴法检测肿瘤细胞运动能力。制作MGC、EC109细胞琼脂悬液,每孔100ul加入24孔板,4℃冷却凝固,测基础半径。分为启膈方小、中、大剂量组、5FU和空白对照组,各组浓度同MTT法。测量各组细胞的运动半径,计算各组细胞的运动速度。明胶酶谱法检测肿瘤细胞分泌MMP-2、MMP-9活性。分组同上,培养24小时后取上清,走聚丙烯酰胺电泳,根据各组条带灰度分析MMP-2、MMP-9活性。
结果:启膈方对MGC、EC109细胞体外增殖有明显的抑制作用,并随着药物浓度增加其抑制作用更加明显。启膈方大剂量组对肿瘤抑制作用最强,加药培养MGC细胞24小时,抑制率为60.0%,72小时抑制率最高为77.1%。启膈方各剂量组、5FU组与对照组之间差异均有显著性(P<0.05)。启膈方大剂量组与5FU组差异无显著性(P>0.05)。培养12小时检测各组细胞运动活性均较弱,各组间差异无显著性。培养24小时后,对照组运动能力明显增强,运动速度明显较启膈方各组快,表现出较强的运动活性。5Fu组细胞运动活性较启膈方组增强,差异有显著性(P<0.05)。启膈方各组之间差异无显著性(P>0.05)。空白对照组细胞分泌MMP-2、MMP-9活性明显较启膈方各组、5FU组增强(P<0.05),其中MMP-2活性增强更明显(P<0.01),启膈方大剂量组活性均较5FU组减弱,差别有显著性(P<0.05),中药各剂量组之间差异无显著性(P>0.05)。
结论:启膈方可抑制MGC、EC109肿瘤细胞的增殖,启膈方大剂量与5FU对肿瘤细胞增殖MMP-2、MMP-9的抑制作用无差异。启膈方可抑制MGC、EC109肿瘤细胞的运动能力,减慢其运动速度。5FU组细胞运动活性较中药组增强。启膈方可抑制MGC、EC109肿瘤细胞分泌MMP-2,MMP-9的活性。
第三部分启膈方对胃癌荷瘤小鼠肺转移的抑制作用
目的:观察启膈方对胃癌荷瘤小鼠肿瘤生长的影响,通过计算肺转移结节,观察启膈方对胃癌荷瘤小鼠肺转移的抑制作用。并比较5FU对胃癌荷瘤小鼠肿瘤生长及肺转移的抑制作用。
方法:取小鼠胃癌FC细胞悬液分别接种在30只615小鼠右腋皮下,每只0.1ml(1×106个细胞)。接种后随机分为,启膈方、5FU、空白对照三组,每组各10只。接种3天后中药组每天灌服启膈方口服液0.2ml(0.3g),化疗组每天腹腔注射5FU0.2ml(200ug),对照组每天灌服生理盐水0.2ml。16天后处死小鼠,剥离肿瘤组织,称瘤重,测肿瘤直径,计算肿瘤体积。取出肺组织用10%中性福尔马林固定,24-48小时后观察,肺组织表面的转移结节,比较各组转移结节数量,计算肺转移抑制率。石蜡包埋切片(厚度4um),常规HE染色,比较各组肺内转移程度。
结果:启膈方组、5FU组瘤重、瘤体积、肿瘤直径明显低于对照组(P<0.01),启膈方组、5FU组之间差异无显著性(P>0.05)。直接计数肺组织表面白色的转移结节,对照组小鼠100%发生肺转移。启膈方组肺转移结节数少于对照组(P<0.01)其中有4例未见转移结节,转移程度明显较对照组轻(P<0.01)。与5FU组比较,启膈方组肺转移结节数、肺转移抑制率也优于5FU组(P<0.05),5FU组中有1例未见转移结节,5FU组与对照组的肺转移结节数、肺内转移程度、肺转移抑制率比较差异均有显著性(P<0.05)。
结论:启膈方可抑制胃癌荷瘤小鼠肿瘤的生长,减少肺转移的发生,减轻肺转移的程度。5FU也可减少肺转移的发生,减轻肺转移的程度,但转移抑制作用不如启膈方。
第四部分启膈方对胃癌转移相关因子的影响
目的:观察启膈方对小鼠胃癌移植瘤的黏附分子E-Cad、蛋白水解酶MMP-2、MMP-9、血管内皮生长因子VEGF、微血管密度MVD表达的影响。检测小鼠血浆GMP-140、TXB2水平,比较各组差别。
方法:分组、造模、灌药同第三部分实验。摘除眼球取血,获得血浆备用,处死小鼠,剥离肿瘤组织。常规用10%中性福尔马林固定,石蜡包埋切片(厚度4um)。用SP免疫组化法检测各组肿瘤组织E-Cad、MMP-2、MMP-9、VEGF、CD34的表达,用放射免疫法检测各组小鼠血浆TXB2水平,用酶联免疫法检测小鼠血浆GMP-140水平。
结果:各组瘤组织均有E-钙粘蛋白不同程度的表达,启膈方组E-cadherin的表达强于空白对照组、5FU组,差异有显著性(P<0.01),5FU组E-cadherin的表达强于空白对照组,差异有显著性(P<0.05)。对照组MVD明显高于启膈方组(P<0.01)和5FU组(P<0.05),启膈方组低于5FU组,差异有显著性(P<0.05)。启膈方组、5FU组的VEGF表达低于对照组,有显著性差异(P<0.05),5FU组与启膈方组比较无显著性差异(P>0.05)。各组瘤组织均有MMP-2、MMP-9不同程度的表达,启膈方组的MMP-2、MMP-9表达弱于空白对照组,有显著性差异(P<0.05),5FU组的表达弱于对照组有显著性差异(P<0.01),与启膈方组比较无显著性差异(P>0.05)。各组小鼠血浆启膈方组、5FU组GMP-140与TXB2含量均明显低于对照组,尤其是血浆GMP-140含量,启膈方组、5FU组与对照组之间有非常显著性差异(P<0.01),启膈方组、5FU组之间差异无显著性。血浆TXB2水平,启膈方组低于5Fu组,差异有显著性(P<0.05)。
结论:启膈方可通过提高黏附分子E-Cad的表达,降低蛋白水解酶MMP-2、MMP-9的表达,降低血管内皮生长因子VEGF表达、降低微血管密度MVD,降低小鼠血浆GMP-140、TXB2水平,而起到抑制转移的作用。
Gastric cancer is a common malignant tumor. As the highest incidence tumor in alimentary system, it’s incidence accounts 62% in alimentary system tumor. The incidence of gastric cancer in our country is high and far higher than western country. About 170 thousand people died of gastric cancer,it takes 50% death number in alimentary system tumor in our country every year. With increasing medical level and applying comprehend treatment,5 year survival rate of gastric cancer has improved,but overall therapeutic effect is still unsatisfactory. The reason is there is non-typical symptom of early gastric cancer, most patients with progression caner when they were diagnosed. The metastasis always causes treatment failure after surgery. It is reported about 40%-60% of patients with gastric cancer recurrence and metastasis. To seek reliable methods to treat gastric cancer metastasis is an urgent problem we are facing.
The primary route of metastasis is blood metastasis, lymphatic metastasis, peritoneum implantation metastasis.Perniciousness of blood route metastasis is the largest dangerous.By the blood circulation gastric cancer-cell can form metastsis in other organ and destroy organ’s function to lead to die. Liver and lung is the most vulnerable organs to the transfer of blood. It is reported liver metastsis incidence is 40-50% and lung metastsis is 40% in gastric cancer.
Chemotherapy is the main therapy after liver and lung metastsis.Although combined with chinese medicine,radiotherapy,operation and immunotherapy median-survival time is only 6-9 months. Therefore, metastasis preventing is key point on cure gastric cancer and important sense to increase survival time. The hot-spots in tumor research are mechanism of tumor metastasis and anti-metastasis medicine. The basic process of tumor metastasis has probably clear. Anti-metastasis medicine concentrate on anti-new-vessels, anti-platelet aggregation, anti-platelet activation, anti-metastasis gene, raising immune function, which most are in experimental stage. Moreover, the metastasis of gastric cancer is multi-factor, multi-step, and multi-link process, while these medicines’single channel and target lead to the role in inhibiting metastasis utterly inadequate. Complex prescription of chinless medicine is reasonable combination based on“planning treatment according to Diagnosis”. It has much complex composition and has many curing function. Thus, we researched anti-metastasis function of Qigefang on the basic process of metastasis and correlated factors.
Qigefang changed from Qigesan in <> which Qigesan was used to treat Dysphasia. While treating patients with gastric cancer and esophageal cancer, we find Qigefang can prolong life span and prevent or delay metastasis of gastric cancer in advanced stage. Qigefang can prevent metastasis to prolong disease free survival date (DFS) used for patients after they were operated. Therefore we think Qigefang has anti-metastasis function on gastric cancer. The research investigates Qigefang’s effective target and effective link on restraining metastasis from process blood route metastasis of gastric cancer.
We observed anti-metastasis function of Qigefang in vitro test and in vivo test. After cultured high-invasion cell line of gastric cancer and esophageal cancer, we observed the influence of Qigefangon on metastasis ability of tumor cell. In vivo test, mice were vaccinated by high-lung metastasis gastric cancer cell line, we observed inhibition ratio of lung metastasis and influence on related metastasis factor.
The research includes four parts:
Part one: Effects of Qigefang on adhesion of human gastric cancer cell MGC and of human esophageal cancer cell EC109
Objective: To observe Qigefang’s effect on homogeneous adhesion between gastric cancer cell line MGC and gastric cancer cell line MGC, between human esophageal cancer cell line EC109 and human esophageal cancer cell line EC109. To detect difference of adhesive ratio, aggregate formation ratio and E-Cadherion expression among Qigefang group, 5FU group and control group by several methods.
Methods: Resused human gastric cancer cell line MGC and human esophageal cancer cell line EC109 were devived randomly into five groups when cell was at logarithmic growth phase. Add Qigefang in Chinese medicine group to make final concentration be 15mg/ml,30mg/ml,60mg/ml and called low-dose group,meta-dose group, macro-dose group; Add 5FU chemothapy group to make final concentration be 50ug/ml;add PBS in control group.To detect adhersion status at 20min,40min,60min of each group by machine method and to compute adhersion ratio. To detect intensity of radiation at 30min,60min by active nucleus and to compute adhersion ratio. To detect formation of aggregate at 2h,4h,6h,12h and to compute formation ratio of aggregate and measure the largest area of aggregate in each group.To measure expression of E-cadherin by flow cytometry.
Results:To detect adhersion ratio of gastric cancer cell MGC and esophageal cancer cell EC109 at 20min,40min,60min by machine mothod ,each dose group of Qigefang was higher than 5FU group and control group.The difference between Qigefang group and 5FU group or control group was significant(P<0.01). The difference between 5FU group and control group was not significant(P>0.05). The difference among each dose group of Qigefang was not significant(P>0.05). By active nucleus to detect adhersion ratio of gastric cancer cell MGC and esophageal cancer cell EC109 at30min,60min ,each dose group of Qigefang was higher than 5FU group and control group.The difference between Qigefang group and 5FU group or control group was significant(P<0.01). The difference between 5FU group and control group was not significant(P>0.05). The difference among each dose group of Qigefang was not significant(P>0.05). The largest area of aggregate of MGC and EC109 in each dose Qigefang group was larger than control group and 5FU group (P<0.05), 5FU group was smaller than control group.
Expression of E-cadherin in each Qigefang was higer than control group and 5FU group by flow cytometry. The difference between 5FU group and control group was not significant(P>0.05), The difference among each dose group of Qigefang was not significant(P>0.05).
Conclusion: Qigefang could raise adhersion ratio,formimg ratio of aggregate of human gastric cancer cell line MGC and human esophageal cancer cell line EC109.Qigefang could raise homogeneous adhersion capability.5FU had not obvious influence about homogeneous adhersion ratio.when 5FU effected tumor cell for a long time,it would retrain aggregate form. Qigefang could raise E-cadherin expression of human gastric cancer cell line MGC and human esophageal cancer cell line EC109. Part two: Effects of Qigefang on cell proliferation,movement,secreting MMP-2 and MMP-9 of of human gastric cancer cell MGC and of human esophageal cancer cell EC109
Objective: To observe that Qigefang had inhibitory action on cell proliferation,movement,secreting MMP-2 and MMP-9 of MGC and EC109 cell line.Compare with 5FU group,found the difference in cytostasis ratio,moving speed,enzyme activity of MMP-2 and MMP-9.
Methods:To detect tumor cell proliferation by MTT mothod,100ul Cell suspension was added in each hole of 96 orifice(each hole 2×105 cell)and tumor cell was divided into Qigefang group,5FU group and control group.Qigefang were added into Qigefang group to make final concentration be 15mg/ml、30mg/ml、60mg/ml and called low-dose group,meta-dose group, macro-dose group. 5FU were added into 5FU group to make final concentration be 50ug/ml. PBS was added into control group.Incubated 24,48,72h in incubaton and added MTT 4h before incubation accomplished.Detect OD value in each time spot and count proliferation inhibition ratio. To detect moving ablity of tumor cell by agar drop mothod.Made agar suspension of MGC,EC109 cell and added 100ul in each hole of 24 orifice to detect basic radius after cooling congelation at 4℃.Divided into Qigefang group,5FU group and control group. Medical concentration of each group were similar to MTT method.To measure movement radius of each group cell and count moving speed of each group cell.To detect MMP-2,MMP-9 activity by gelatinase zymography .Groups were same as above.To obtain supernatant after cultured 24h and made polyacrylamide gel electrophoresis.To analyze MMP-2,MMP-9 activty based on strap gray scale.
Results: Qigefang had obviously depressant effect on vitro-proliferation of MGC,EC109 cell.It’s depressant fuction became more obviously with raising medicine density. Macro-dose of chinese medicine had the strongest depressant effect on tumor cell.Inhibition ratio had reach 60.0% .The highest depressant ratio was 77.1% after cultured 72h with Chinese medicine. The difference among each-dose of Chinese medicine group ,5FU group and control group was all significant ( P<0.05 ) . The difference between Macro-dose of Chinese medicine group and 5FU group was not significant(P>0.05).To detect moving action of each group cell was all poor when cultured at 12h. Moving ablity of control group became strong obviously and moving speed was faster than Qigefang after cultured 24h.Control group showed strong locomotor activity. locomotor activity of 5FU group was also stronger than Qigefang group, The difference was significant(P<0.05). The difference among each dose group of Qigefang was not significant(P>0.05).Control group cell secretion of MMP-2,MMP-9 was more than Qigefang group and 5FU group(P<0.05).MMP-2 was stronger obviously(P<0.01).Activity of MMP-2 and MMP-9 in Qigefang group was waker than 5FU group. The difference was significant(P<0.05)and the difference between each-dose of Chinese medicine was not significant(P>0.05).
Conclusion: Qigefang could suppress reproductive activity of MGC and EC109 cell, Macro-dose compare with 5FU the difference was not significant. Qigefang could suppress moving ablity of MGC and EC109 cell and slow down moving speed. Cell locomotor activity of 5FU group was stronger Chinese medicine group.Qigefang could suppress activity of MMP-2 and MMP-9 which tumor secreted.Enzymatic activity of 5FU was stronger than Qigefang group.
Part three: Inhibiting effects of Qigefang on lung metastsis of mice with gastric bearing cancer
Objective: To observe Qigefang effect on tumor growing of mice with gastric bearing cancer. To observe Qigefang depressant function on pulmonary metastsis of mice with gastric bearing cancer by counting pulmonary metastsis nodus and to compare 5FU depressant function on pulmonary metastsis of gastric bearing cancer of mice.
Mothods: To obtain cell suspension of gastric cancer to vaccinate at right armpit hypo of 30mice 615, a mouse 0.1ml(1×10 6cell).Divided into three groups randomly------Qigefang group, 5FU group,control group.Each group was 10 mice. After vaccinated 3 days,the mice in Chinese medicine group were drenched Qigefang fluid 0.2ml(0.3g)each day. the mouse in chemotherapy group were injected 5FU 0.2ml(200ug) in abdominal cavity. the mouse in control group were drenched isotonic Na chloride 0.2ml.After 16 days killed the mouse,stripped the tumor architecture,weighed tumor,measuse tumor diameter,compute gross tumor volume.Dislodged lung architecture and fixed by 10% neutro formalin.To observse metastasis on lung architecture,compared quantity of metastasis nodus,computer depressant ratio of pulmonary metastsis.Paraffin imbedding,cut sheet(thickness 4um),HE drum dyeing commonly,compare metastasis degree in lung with each group.
Results: Tumor weight ,tumor diameter and tumor volume of Chinese medicine group and chemothapy group were obviously lower than control group(P<0.01).The difference between Chinese group and chemotherapy group was not significant(P>0.05).Mouse of control group 100% happened lung metastsis by directly counted white metastsis nodus on lung surface.Number of metastsis nodus on lung were less than control group’s and metastsis degree were less than control group obviously(P<0.01).There were 4 mice no metastsis nodus(P<0.01 ).Compared with chemotherapy group, metastsis nodus on lung and inhibition ratio of lung of Chinese medicine group was better than chemotherapy and there was difference of metastsis degree between Chinese medicine group and control group . There was a mouse no metastsis nodus in chemotherapy group.The difference of number of metastsis nodus,lung metastsis degree and inhibition ratio of lung metastsis between chemotherapy group and control group was significant(P<0.05).
Conclusion: Qigefang could inhibit tumor growing of mice model of gastric cancer,decrease pulmonary metastsis,lessen lung metastsis degree.5FU also could decrease pulmonary metastsis and lessen lung metastsis degree,but inhibitory action on metastsis inferior to Qigefang.
Part four: Effects of Qigefang on gastric cancer metastsis correlation factor
Objective: To observe Qigefang effect on adhesion molecule E-cad ,Proteo lytic enzyme MMP-2,MMP-9,blood vessel endothelial growth factor VEGF,microvessel density MVD of transplantation tumor of mouse gastric cancer.To detect blood plasm level of GMP-140, GMP-140 of mice . and find difference among groups.
Mothods: Grouping,making model,pouring medicine were similar to experiment in part three .Removal eye globe to obtain blood,gained blood plasm for pre-emergency,killed the mouse,stripped tumor architecture. 10% neutro formalin fixed. Paraffin imbedding,cut sheet(thickness 4um).To detect expression of E-Cad、MMP-2、MMP-9、VEGF、MVD on tumor of each group by SP immunohistochemistry.To detect GMP-140 level of mouse blood plasm by radio-immunity.To detect GMP-140 level of mouse blood plasm by euzymelinked immunosorbent assay.
Results: There were different degree expression of E-cad on tumor architecture of each group.Expression of E-cad of Chinese medicine group was stronger than control group and 5FU group. The difference was significant(P<0.01). The difference between control group and 5FU group was also significant(P<0.05).MVD of control group was obviously higher than Chinese group and 5FU group(P<0.05). The difference between Chinese medicine group and 5FU group was significant(P<0.05). Expression of VEGF of Chinese medicine group and 5FU group was lower than control group. The difference was significant(P<0.05). The difference between Chinese medicine group and 5FU group was not significant(P>0.05). There was different degree expression of MMP-2,MMP-9 on tumor architecture of each group. Expression of MMP-2,MMP-9 of Chinese medicine group was lower than contrl group, The difference was significant(P<0.05).Expression of MMP-2,MMP-9 of 5FU group was also lower than control group, The difference was significant(P<0.05). The difference between Chinese medicine group and 5FU group was not significant(P>0.05). level of GMP-140 and TXB2 of mouse blood plasm Chinese medicine group and 5FU group was lower than control group obviously, especially level of blood plasm GMP-140, The difference among Chinese medicine group ,5FU group and control group was highly significant(P<0.01),the difference between Chinese medicine group and 5FU group was not significant. level of blood plasm TXB2, the difference between Chinese medicine group and 5FU group was significant(P<0.05).
Conclusion:Qigefang can raise expression of adhesion molecule E-cad, depress expression of proteolytic enzyme MMP-2 and MMP-9, depress expression of blood vessel endothelial growth factor VEGF, depress microvessel density MVD, depress GMP-140,TXB2 level of mouse blood plasm.Therefor,Qigefang has inhibiting effection on metastsis.
胃癌转移的主要途径为血道转移、淋巴道转移、腹膜种植转移。其中血道转移的危害最大,通过血液循环系统胃癌细胞可在其他脏器形成转移灶,破坏脏器功能最终导致死亡。肝、肺是胃癌血道转移最易发生的器官,文献报道胃癌肝转移的发生率为40-50%,肺转移的发生率为40%左右。肝、肺转移后多以全身化疗为主,尽管结合中药、手术、放疗、免疫治疗,其中位生存时间也仅为6-9月。由此可见,防止转移的发生是治疗胃癌的重要课题,对延长胃癌生存期有重要意义。
肿瘤转移的机制及抗肿瘤转移药物的研究是肿瘤研究的热点,肿瘤转移的基本过程已大概清楚,抗肿瘤转移药物集中在抗新生血管、抗血小板聚集、抗血小板活化、抑制转移基因表达、提高免疫等方面,多数处于实验研究阶段,而且这些药物作用途径、作用靶点单一,对于多步骤、多环节、多因素的胃癌转移过程,其抑制转移的作用显得杯水车薪。中药复方是在辩证论治的基础上的合理组合,它含有多种复杂成分,有多种治疗作用。由此,我们从转移的基本过程及相关因素,研究中药复方启膈方的抗转移作用。
启膈方是根据《医学心悟》中治疗噎嗝的启膈散化裁而成,临床用于治疗胃癌、食管癌患者。发现启膈方用于晚期胃癌患者可延长生存时间,防止、延缓转移灶的出现;用于胃癌术后患者可防止转移,延长无病生存时间。于是我们考虑该方有抗胃癌转移的作用,本研究从胃癌血道转移的过程入手,探讨启膈方抑制转移的作用环节及作用靶点。
本课题从体外实验、体内实验两个方面研究启膈方的抗转移作用。体外培养高侵袭胃癌、食管癌瘤株,启膈方干预后,观察对肿瘤细胞转移能力的影响。体内动物实验,用高肺转移小鼠胃癌瘤株FC接种615小鼠后灌服启膈方,观察肺转移抑制率及对相关转移因子的影响。
本课题分为四部分完成:
第一部分启膈方对人胃癌细胞MGC、人食管癌细胞EC109黏附的影响
目的:观察启膈方对人胃癌细胞株MGC与人胃癌细胞株MGC之间、人食管癌细胞株EC109与人食管癌细胞株EC109之间同质黏附的影响。用不同方法检测比较启膈方组、5FU组、空白对照组的黏附率、聚集体形成率及E-钙黏蛋白表达的差别。
方法:复苏人胃癌细胞株MGC、人食管癌细胞株EC109,待细胞呈对数生长期时,随机分为5组。启膈组加入启膈方,使其终浓度为15mg/ml、30mg/ml、60mg/ml,定为启膈方小、中、大剂量组。5FU组加入5FU,使其终浓度为50ug/ml,对照组加入PBS。用机械法检测各组细胞20、40、60分钟黏附情况,计算黏附率。用3-H-Tdr标记的放射性核素法检测各组细胞30、60分钟时液闪仪测定的放射强度,计算黏附率。用机械法检测各组细胞2、4、6、12小时聚集体形成情况,计算聚集体形成率,并测量各组细胞聚集体的最大面积。流式细胞术测定各组E-钙黏连蛋白的相对表达量。
结果:机械法检测MGC胃癌细胞,EC109食管癌细胞在20min、40min、60min的黏附率。启膈方各剂量组的黏附率均高于5FU组、空白对照组。启膈方各剂量组与空白对照组、5FU组之间差异有显著性(P<0.01),空白对照组、5FU组之间差异无显著性(P>0.05),启膈方各剂量组之间差异无显著性(P>0.05)。放射核素法检测MGC胃癌细胞、EC109食管癌细胞30分钟、60分钟黏附率,启膈方各剂量组均高于5FU组、空白对照组,启膈方各剂量组与空白对照组、5Fu组之间差异有显著性(P<0.05),空白对照组、5FU组之间差异无显著性(P>0.05),启膈方各剂量组之间差异无显著性(P>0.05)。MGC细胞、EC109细胞启膈方各剂量组的各时间点聚集体形成率、聚集体面积均大于空白对照组、5FU组(P<0.05),5FU组小于空白对照组(P<0.05),启膈方各剂量组之间差异无显著性(P>0.05)。流式细胞仪检测E-钙黏蛋白含量启膈方各剂量组均高于空白对照组、5FU组(P<0.05),空白对照组与5FU组之间差异无显著性(P>0.05),启膈方各剂量组之间差异无显著性(P>0.05)。
结论:启膈方可提高MGC人胃癌细胞、EC109人食管癌细胞的黏附率、聚集体形成率、提高了同质黏附能力,5FU对同质黏附率无明显影响,长时间作用于肿瘤细胞后影响聚集体的形成。启膈方可提高MGC人胃癌细胞、EC109人食管癌细胞E-Cadherin蛋白的表达。
第二部分启膈方对人胃癌细胞MGC、人食管癌细胞EC109增殖、运动、分泌MMP-2、MMP-9的影响
目的:观察启膈方对MGC、EC109细胞增殖能力、运动能力、分泌MMP-2、MMP-9的抑制作用,比较5FU组MGC、EC109细胞增殖抑制率,运动速度、MMP-2、MMP-9活性的不同。
方法:MTT法检测肿瘤细胞的增殖。在96孔板上每孔加入细胞悬液100ul,细胞数为2×103,分为启膈方、5FU和空白对照组,启膈方组分别加入启膈方,使其终浓度为15mg/ml、30mg/ml、60mg/ml定为小、中、大剂量组,5FU组加入5FU注射液,使其终浓度为50ug/ml,在空白对照组加入PBS。培养箱中孵育24、48、72h,培养结束前4小时加入MTT,检测各时间点的OD值,计算增殖抑制率。琼脂滴法检测肿瘤细胞运动能力。制作MGC、EC109细胞琼脂悬液,每孔100ul加入24孔板,4℃冷却凝固,测基础半径。分为启膈方小、中、大剂量组、5FU和空白对照组,各组浓度同MTT法。测量各组细胞的运动半径,计算各组细胞的运动速度。明胶酶谱法检测肿瘤细胞分泌MMP-2、MMP-9活性。分组同上,培养24小时后取上清,走聚丙烯酰胺电泳,根据各组条带灰度分析MMP-2、MMP-9活性。
结果:启膈方对MGC、EC109细胞体外增殖有明显的抑制作用,并随着药物浓度增加其抑制作用更加明显。启膈方大剂量组对肿瘤抑制作用最强,加药培养MGC细胞24小时,抑制率为60.0%,72小时抑制率最高为77.1%。启膈方各剂量组、5FU组与对照组之间差异均有显著性(P<0.05)。启膈方大剂量组与5FU组差异无显著性(P>0.05)。培养12小时检测各组细胞运动活性均较弱,各组间差异无显著性。培养24小时后,对照组运动能力明显增强,运动速度明显较启膈方各组快,表现出较强的运动活性。5Fu组细胞运动活性较启膈方组增强,差异有显著性(P<0.05)。启膈方各组之间差异无显著性(P>0.05)。空白对照组细胞分泌MMP-2、MMP-9活性明显较启膈方各组、5FU组增强(P<0.05),其中MMP-2活性增强更明显(P<0.01),启膈方大剂量组活性均较5FU组减弱,差别有显著性(P<0.05),中药各剂量组之间差异无显著性(P>0.05)。
结论:启膈方可抑制MGC、EC109肿瘤细胞的增殖,启膈方大剂量与5FU对肿瘤细胞增殖MMP-2、MMP-9的抑制作用无差异。启膈方可抑制MGC、EC109肿瘤细胞的运动能力,减慢其运动速度。5FU组细胞运动活性较中药组增强。启膈方可抑制MGC、EC109肿瘤细胞分泌MMP-2,MMP-9的活性。
第三部分启膈方对胃癌荷瘤小鼠肺转移的抑制作用
目的:观察启膈方对胃癌荷瘤小鼠肿瘤生长的影响,通过计算肺转移结节,观察启膈方对胃癌荷瘤小鼠肺转移的抑制作用。并比较5FU对胃癌荷瘤小鼠肿瘤生长及肺转移的抑制作用。
方法:取小鼠胃癌FC细胞悬液分别接种在30只615小鼠右腋皮下,每只0.1ml(1×106个细胞)。接种后随机分为,启膈方、5FU、空白对照三组,每组各10只。接种3天后中药组每天灌服启膈方口服液0.2ml(0.3g),化疗组每天腹腔注射5FU0.2ml(200ug),对照组每天灌服生理盐水0.2ml。16天后处死小鼠,剥离肿瘤组织,称瘤重,测肿瘤直径,计算肿瘤体积。取出肺组织用10%中性福尔马林固定,24-48小时后观察,肺组织表面的转移结节,比较各组转移结节数量,计算肺转移抑制率。石蜡包埋切片(厚度4um),常规HE染色,比较各组肺内转移程度。
结果:启膈方组、5FU组瘤重、瘤体积、肿瘤直径明显低于对照组(P<0.01),启膈方组、5FU组之间差异无显著性(P>0.05)。直接计数肺组织表面白色的转移结节,对照组小鼠100%发生肺转移。启膈方组肺转移结节数少于对照组(P<0.01)其中有4例未见转移结节,转移程度明显较对照组轻(P<0.01)。与5FU组比较,启膈方组肺转移结节数、肺转移抑制率也优于5FU组(P<0.05),5FU组中有1例未见转移结节,5FU组与对照组的肺转移结节数、肺内转移程度、肺转移抑制率比较差异均有显著性(P<0.05)。
结论:启膈方可抑制胃癌荷瘤小鼠肿瘤的生长,减少肺转移的发生,减轻肺转移的程度。5FU也可减少肺转移的发生,减轻肺转移的程度,但转移抑制作用不如启膈方。
第四部分启膈方对胃癌转移相关因子的影响
目的:观察启膈方对小鼠胃癌移植瘤的黏附分子E-Cad、蛋白水解酶MMP-2、MMP-9、血管内皮生长因子VEGF、微血管密度MVD表达的影响。检测小鼠血浆GMP-140、TXB2水平,比较各组差别。
方法:分组、造模、灌药同第三部分实验。摘除眼球取血,获得血浆备用,处死小鼠,剥离肿瘤组织。常规用10%中性福尔马林固定,石蜡包埋切片(厚度4um)。用SP免疫组化法检测各组肿瘤组织E-Cad、MMP-2、MMP-9、VEGF、CD34的表达,用放射免疫法检测各组小鼠血浆TXB2水平,用酶联免疫法检测小鼠血浆GMP-140水平。
结果:各组瘤组织均有E-钙粘蛋白不同程度的表达,启膈方组E-cadherin的表达强于空白对照组、5FU组,差异有显著性(P<0.01),5FU组E-cadherin的表达强于空白对照组,差异有显著性(P<0.05)。对照组MVD明显高于启膈方组(P<0.01)和5FU组(P<0.05),启膈方组低于5FU组,差异有显著性(P<0.05)。启膈方组、5FU组的VEGF表达低于对照组,有显著性差异(P<0.05),5FU组与启膈方组比较无显著性差异(P>0.05)。各组瘤组织均有MMP-2、MMP-9不同程度的表达,启膈方组的MMP-2、MMP-9表达弱于空白对照组,有显著性差异(P<0.05),5FU组的表达弱于对照组有显著性差异(P<0.01),与启膈方组比较无显著性差异(P>0.05)。各组小鼠血浆启膈方组、5FU组GMP-140与TXB2含量均明显低于对照组,尤其是血浆GMP-140含量,启膈方组、5FU组与对照组之间有非常显著性差异(P<0.01),启膈方组、5FU组之间差异无显著性。血浆TXB2水平,启膈方组低于5Fu组,差异有显著性(P<0.05)。
结论:启膈方可通过提高黏附分子E-Cad的表达,降低蛋白水解酶MMP-2、MMP-9的表达,降低血管内皮生长因子VEGF表达、降低微血管密度MVD,降低小鼠血浆GMP-140、TXB2水平,而起到抑制转移的作用。
Gastric cancer is a common malignant tumor. As the highest incidence tumor in alimentary system, it’s incidence accounts 62% in alimentary system tumor. The incidence of gastric cancer in our country is high and far higher than western country. About 170 thousand people died of gastric cancer,it takes 50% death number in alimentary system tumor in our country every year. With increasing medical level and applying comprehend treatment,5 year survival rate of gastric cancer has improved,but overall therapeutic effect is still unsatisfactory. The reason is there is non-typical symptom of early gastric cancer, most patients with progression caner when they were diagnosed. The metastasis always causes treatment failure after surgery. It is reported about 40%-60% of patients with gastric cancer recurrence and metastasis. To seek reliable methods to treat gastric cancer metastasis is an urgent problem we are facing.
The primary route of metastasis is blood metastasis, lymphatic metastasis, peritoneum implantation metastasis.Perniciousness of blood route metastasis is the largest dangerous.By the blood circulation gastric cancer-cell can form metastsis in other organ and destroy organ’s function to lead to die. Liver and lung is the most vulnerable organs to the transfer of blood. It is reported liver metastsis incidence is 40-50% and lung metastsis is 40% in gastric cancer.
Chemotherapy is the main therapy after liver and lung metastsis.Although combined with chinese medicine,radiotherapy,operation and immunotherapy median-survival time is only 6-9 months. Therefore, metastasis preventing is key point on cure gastric cancer and important sense to increase survival time. The hot-spots in tumor research are mechanism of tumor metastasis and anti-metastasis medicine. The basic process of tumor metastasis has probably clear. Anti-metastasis medicine concentrate on anti-new-vessels, anti-platelet aggregation, anti-platelet activation, anti-metastasis gene, raising immune function, which most are in experimental stage. Moreover, the metastasis of gastric cancer is multi-factor, multi-step, and multi-link process, while these medicines’single channel and target lead to the role in inhibiting metastasis utterly inadequate. Complex prescription of chinless medicine is reasonable combination based on“planning treatment according to Diagnosis”. It has much complex composition and has many curing function. Thus, we researched anti-metastasis function of Qigefang on the basic process of metastasis and correlated factors.
Qigefang changed from Qigesan in <
We observed anti-metastasis function of Qigefang in vitro test and in vivo test. After cultured high-invasion cell line of gastric cancer and esophageal cancer, we observed the influence of Qigefangon on metastasis ability of tumor cell. In vivo test, mice were vaccinated by high-lung metastasis gastric cancer cell line, we observed inhibition ratio of lung metastasis and influence on related metastasis factor.
The research includes four parts:
Part one: Effects of Qigefang on adhesion of human gastric cancer cell MGC and of human esophageal cancer cell EC109
Objective: To observe Qigefang’s effect on homogeneous adhesion between gastric cancer cell line MGC and gastric cancer cell line MGC, between human esophageal cancer cell line EC109 and human esophageal cancer cell line EC109. To detect difference of adhesive ratio, aggregate formation ratio and E-Cadherion expression among Qigefang group, 5FU group and control group by several methods.
Methods: Resused human gastric cancer cell line MGC and human esophageal cancer cell line EC109 were devived randomly into five groups when cell was at logarithmic growth phase. Add Qigefang in Chinese medicine group to make final concentration be 15mg/ml,30mg/ml,60mg/ml and called low-dose group,meta-dose group, macro-dose group; Add 5FU chemothapy group to make final concentration be 50ug/ml;add PBS in control group.To detect adhersion status at 20min,40min,60min of each group by machine method and to compute adhersion ratio. To detect intensity of radiation at 30min,60min by active nucleus and to compute adhersion ratio. To detect formation of aggregate at 2h,4h,6h,12h and to compute formation ratio of aggregate and measure the largest area of aggregate in each group.To measure expression of E-cadherin by flow cytometry.
Results:To detect adhersion ratio of gastric cancer cell MGC and esophageal cancer cell EC109 at 20min,40min,60min by machine mothod ,each dose group of Qigefang was higher than 5FU group and control group.The difference between Qigefang group and 5FU group or control group was significant(P<0.01). The difference between 5FU group and control group was not significant(P>0.05). The difference among each dose group of Qigefang was not significant(P>0.05). By active nucleus to detect adhersion ratio of gastric cancer cell MGC and esophageal cancer cell EC109 at30min,60min ,each dose group of Qigefang was higher than 5FU group and control group.The difference between Qigefang group and 5FU group or control group was significant(P<0.01). The difference between 5FU group and control group was not significant(P>0.05). The difference among each dose group of Qigefang was not significant(P>0.05). The largest area of aggregate of MGC and EC109 in each dose Qigefang group was larger than control group and 5FU group (P<0.05), 5FU group was smaller than control group.
Expression of E-cadherin in each Qigefang was higer than control group and 5FU group by flow cytometry. The difference between 5FU group and control group was not significant(P>0.05), The difference among each dose group of Qigefang was not significant(P>0.05).
Conclusion: Qigefang could raise adhersion ratio,formimg ratio of aggregate of human gastric cancer cell line MGC and human esophageal cancer cell line EC109.Qigefang could raise homogeneous adhersion capability.5FU had not obvious influence about homogeneous adhersion ratio.when 5FU effected tumor cell for a long time,it would retrain aggregate form. Qigefang could raise E-cadherin expression of human gastric cancer cell line MGC and human esophageal cancer cell line EC109. Part two: Effects of Qigefang on cell proliferation,movement,secreting MMP-2 and MMP-9 of of human gastric cancer cell MGC and of human esophageal cancer cell EC109
Objective: To observe that Qigefang had inhibitory action on cell proliferation,movement,secreting MMP-2 and MMP-9 of MGC and EC109 cell line.Compare with 5FU group,found the difference in cytostasis ratio,moving speed,enzyme activity of MMP-2 and MMP-9.
Methods:To detect tumor cell proliferation by MTT mothod,100ul Cell suspension was added in each hole of 96 orifice(each hole 2×105 cell)and tumor cell was divided into Qigefang group,5FU group and control group.Qigefang were added into Qigefang group to make final concentration be 15mg/ml、30mg/ml、60mg/ml and called low-dose group,meta-dose group, macro-dose group. 5FU were added into 5FU group to make final concentration be 50ug/ml. PBS was added into control group.Incubated 24,48,72h in incubaton and added MTT 4h before incubation accomplished.Detect OD value in each time spot and count proliferation inhibition ratio. To detect moving ablity of tumor cell by agar drop mothod.Made agar suspension of MGC,EC109 cell and added 100ul in each hole of 24 orifice to detect basic radius after cooling congelation at 4℃.Divided into Qigefang group,5FU group and control group. Medical concentration of each group were similar to MTT method.To measure movement radius of each group cell and count moving speed of each group cell.To detect MMP-2,MMP-9 activity by gelatinase zymography .Groups were same as above.To obtain supernatant after cultured 24h and made polyacrylamide gel electrophoresis.To analyze MMP-2,MMP-9 activty based on strap gray scale.
Results: Qigefang had obviously depressant effect on vitro-proliferation of MGC,EC109 cell.It’s depressant fuction became more obviously with raising medicine density. Macro-dose of chinese medicine had the strongest depressant effect on tumor cell.Inhibition ratio had reach 60.0% .The highest depressant ratio was 77.1% after cultured 72h with Chinese medicine. The difference among each-dose of Chinese medicine group ,5FU group and control group was all significant ( P<0.05 ) . The difference between Macro-dose of Chinese medicine group and 5FU group was not significant(P>0.05).To detect moving action of each group cell was all poor when cultured at 12h. Moving ablity of control group became strong obviously and moving speed was faster than Qigefang after cultured 24h.Control group showed strong locomotor activity. locomotor activity of 5FU group was also stronger than Qigefang group, The difference was significant(P<0.05). The difference among each dose group of Qigefang was not significant(P>0.05).Control group cell secretion of MMP-2,MMP-9 was more than Qigefang group and 5FU group(P<0.05).MMP-2 was stronger obviously(P<0.01).Activity of MMP-2 and MMP-9 in Qigefang group was waker than 5FU group. The difference was significant(P<0.05)and the difference between each-dose of Chinese medicine was not significant(P>0.05).
Conclusion: Qigefang could suppress reproductive activity of MGC and EC109 cell, Macro-dose compare with 5FU the difference was not significant. Qigefang could suppress moving ablity of MGC and EC109 cell and slow down moving speed. Cell locomotor activity of 5FU group was stronger Chinese medicine group.Qigefang could suppress activity of MMP-2 and MMP-9 which tumor secreted.Enzymatic activity of 5FU was stronger than Qigefang group.
Part three: Inhibiting effects of Qigefang on lung metastsis of mice with gastric bearing cancer
Objective: To observe Qigefang effect on tumor growing of mice with gastric bearing cancer. To observe Qigefang depressant function on pulmonary metastsis of mice with gastric bearing cancer by counting pulmonary metastsis nodus and to compare 5FU depressant function on pulmonary metastsis of gastric bearing cancer of mice.
Mothods: To obtain cell suspension of gastric cancer to vaccinate at right armpit hypo of 30mice 615, a mouse 0.1ml(1×10 6cell).Divided into three groups randomly------Qigefang group, 5FU group,control group.Each group was 10 mice. After vaccinated 3 days,the mice in Chinese medicine group were drenched Qigefang fluid 0.2ml(0.3g)each day. the mouse in chemotherapy group were injected 5FU 0.2ml(200ug) in abdominal cavity. the mouse in control group were drenched isotonic Na chloride 0.2ml.After 16 days killed the mouse,stripped the tumor architecture,weighed tumor,measuse tumor diameter,compute gross tumor volume.Dislodged lung architecture and fixed by 10% neutro formalin.To observse metastasis on lung architecture,compared quantity of metastasis nodus,computer depressant ratio of pulmonary metastsis.Paraffin imbedding,cut sheet(thickness 4um),HE drum dyeing commonly,compare metastasis degree in lung with each group.
Results: Tumor weight ,tumor diameter and tumor volume of Chinese medicine group and chemothapy group were obviously lower than control group(P<0.01).The difference between Chinese group and chemotherapy group was not significant(P>0.05).Mouse of control group 100% happened lung metastsis by directly counted white metastsis nodus on lung surface.Number of metastsis nodus on lung were less than control group’s and metastsis degree were less than control group obviously(P<0.01).There were 4 mice no metastsis nodus(P<0.01 ).Compared with chemotherapy group, metastsis nodus on lung and inhibition ratio of lung of Chinese medicine group was better than chemotherapy and there was difference of metastsis degree between Chinese medicine group and control group . There was a mouse no metastsis nodus in chemotherapy group.The difference of number of metastsis nodus,lung metastsis degree and inhibition ratio of lung metastsis between chemotherapy group and control group was significant(P<0.05).
Conclusion: Qigefang could inhibit tumor growing of mice model of gastric cancer,decrease pulmonary metastsis,lessen lung metastsis degree.5FU also could decrease pulmonary metastsis and lessen lung metastsis degree,but inhibitory action on metastsis inferior to Qigefang.
Part four: Effects of Qigefang on gastric cancer metastsis correlation factor
Objective: To observe Qigefang effect on adhesion molecule E-cad ,Proteo lytic enzyme MMP-2,MMP-9,blood vessel endothelial growth factor VEGF,microvessel density MVD of transplantation tumor of mouse gastric cancer.To detect blood plasm level of GMP-140, GMP-140 of mice . and find difference among groups.
Mothods: Grouping,making model,pouring medicine were similar to experiment in part three .Removal eye globe to obtain blood,gained blood plasm for pre-emergency,killed the mouse,stripped tumor architecture. 10% neutro formalin fixed. Paraffin imbedding,cut sheet(thickness 4um).To detect expression of E-Cad、MMP-2、MMP-9、VEGF、MVD on tumor of each group by SP immunohistochemistry.To detect GMP-140 level of mouse blood plasm by radio-immunity.To detect GMP-140 level of mouse blood plasm by euzymelinked immunosorbent assay.
Results: There were different degree expression of E-cad on tumor architecture of each group.Expression of E-cad of Chinese medicine group was stronger than control group and 5FU group. The difference was significant(P<0.01). The difference between control group and 5FU group was also significant(P<0.05).MVD of control group was obviously higher than Chinese group and 5FU group(P<0.05). The difference between Chinese medicine group and 5FU group was significant(P<0.05). Expression of VEGF of Chinese medicine group and 5FU group was lower than control group. The difference was significant(P<0.05). The difference between Chinese medicine group and 5FU group was not significant(P>0.05). There was different degree expression of MMP-2,MMP-9 on tumor architecture of each group. Expression of MMP-2,MMP-9 of Chinese medicine group was lower than contrl group, The difference was significant(P<0.05).Expression of MMP-2,MMP-9 of 5FU group was also lower than control group, The difference was significant(P<0.05). The difference between Chinese medicine group and 5FU group was not significant(P>0.05). level of GMP-140 and TXB2 of mouse blood plasm Chinese medicine group and 5FU group was lower than control group obviously, especially level of blood plasm GMP-140, The difference among Chinese medicine group ,5FU group and control group was highly significant(P<0.01),the difference between Chinese medicine group and 5FU group was not significant. level of blood plasm TXB2, the difference between Chinese medicine group and 5FU group was significant(P<0.05).
Conclusion:Qigefang can raise expression of adhesion molecule E-cad, depress expression of proteolytic enzyme MMP-2 and MMP-9, depress expression of blood vessel endothelial growth factor VEGF, depress microvessel density MVD, depress GMP-140,TXB2 level of mouse blood plasm.Therefor,Qigefang has inhibiting effection on metastsis.
引文
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