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The Research of Huangmo Alkali-dissolved Polysaccharides Anti-tumor Effect and Regulating Expression of MMPs/TIMPs

Author: LiuKun
Tutor: MaYan
School: Jilin University
Course: Biochemistry and Molecular Biology
Keywords: alkali-dissolved polysaccharides of Huangmo anti-tumor MMP-9 MMP-2 TIMP-1
CLC: R285
Type: Master's thesis
Year: 2006
Downloads: 101
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Department of Biochemistry and Molecular Biology, Basic MedicalScience School of Jilin University. Supervisor : Ma Yan.1. BackgroundPolysaccharides as an important component of organism have severalbiologic activities,such as improving immunity, anti-tumor , anti-virus and soon , nearly without any toxicity. Polysaccharides of Huangmo is immunologicregulator and has significant effect of anti-tumor on Sarcoma 180 etc in vivo andvitro. However, besides of regulating immunity, there were few report aboutstudy of its other anti-tumor mechanism. The matrix metalloproteinases( MMPs ) are main enzymes to degrade extracellular matrix. The type Ⅳcollgenases, consisting of MMP-9(92 kDa) and MMP-2(72 kDa), are the mainmember of MMPs family, and are related to degradation of the type Ⅳcollagens which is main component of the basement memberane. Tissueinhibitor of metalloproteinase-1(TIMP-1) are natural inhibitor of MMP-9.Balance of MMPs and TIMPs is thought as play an important role in the processof tumor invasion and metastasis.This study aimed to explore the anti-tumor effect of alkali-dissolvedpolysaccharides of Huangmo (Fb) in vivo and vitro and correlation of Fb andMMPs/TIMPs expression in tumor growth and metastasis, making use ofmethods of making animal model , cell culture , gelatin zymography,immunohistochemistry and RT-PCR.2. Study about anti-tumor effect of Fb.2.1 Method :2.1.1 Clear class ICR mice were planted liver cancer H22 and divided randomlyinto 5 groups: Fb low dose group (20 mg·kg-1), Fb middle dose group (40mg·kg-1), Fb high dose group (80 mg·kg-1), the NS group as the negative controland the CTX (30 mg·kg-1) group as the positive control. After been given drugs10 days routinely , all mice were killed and taken tumor tissue out. Weighttumor and detect the inhibition rate(IR).2.1.2 Glioma C6 cell lines, prostate carcinoma PC-3M cell lines and matrix neckcarcinoma Hela cell lines were cultured for 48 hours in condition of giving Fb500μg·ml-1﹑250μg·ml-1﹑125μg·ml-1﹑62.5μg·ml-1 and 30μg·ml-1。The MTT assay were used to observe the inhibitory effects of three cell lines.2.2 Result:2.2.1 Average IR of Fb low﹑middle and high dose group were 38.7%﹑51.2%and 58.3%(P<0.05). The life quality of Fb groups mice were superior to controlgroups.2.2.2 With the increase of concentration, the inhibition rate of Fb to C6 cellsincreased from 16.17~57.04 %(P<0.01). Fb had not apparent inhibitory effectson PC-3M and Hela cells(P >0.05).2.3 Conclusion: The results show that Fb can inhibit tumor growth nearlywithout toxicity in vivo and vitro.3. Study about Fb effect on MMP-2﹑MMP-9 and TIMP-1 expression intumor tissue.3.1 Methods:Gelatin zymography was used to detect activities of MMP-2 and MMP-9 inliver cancer H22;SP immunohistochemistry was used to detect proteinexpression of MMP-2﹑MMP-9 and TIMP-1 in liver cancer H22;RT-PCR wasused to detect mRNA expression of MMP-9 and TIMP-1 in liver cancer H22.3.2 Results3.2.1 Result of gelatin zymography show that compareing with control groups,activities of MMP-2 were decreased significantly(P<0.05)and activities ofMMP-9 had not apparent effects(P >0.05)in Fb groups.3.2.2 MMP-2 and MMP-9 positive protein expression in all groups had noapparent difference.The positive protein expression of TIMP-1 in Fb low,middle and high groups were respective 44.4%﹑70% and 84.5%, which wereover-expression compareing with the NS group (41.7%) in SPimmunohistochemistry.3.2.3 TIMP-1 mRNA expression in Fb groups was higher than negative andpositive control group(P<0.05). MMP-9 mRNA expression in all groups had noapparent difference (P >0.05).3.3 Conclusion: All results show that Fb can decrease activity and proteinexpression of MMP-2,and regulate protein and mRNA expression of MMP-9and TIMP-1and repair broken balance of MMP-9/TIMP-1in tumor tissue.In conclusion, alkali-dissolved polysaccharide of Huangmo has strongactivity of anti-tumor, with security and innocuity. Its functional mechanism ofanti-tumor growth and metastasis probably correlates with its effect onregulating activity, protein expression and mRNA expression of MMPs andTIMPs.

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