| Brain glioma is one of the most difficultly cured tumor in neurosurgery. Its high malignancy, quickly growth, powerful invasion lead to the difficulty in operation. In spite of using radiotherapy and chemotherapeutics after operation, tumor cells still have recurrence quickly. Its mortality is high and brain glioma has become a severe threat to people, s health. In recent years, the appearance and development of gene and stem cell therapy provides a broad development space.MSC has a potency of multi-directional differentiation and can be induced to form cartilage cell,fat cell,nerve cell and so on. Its good quality of convenient use,abundant resource,simple cultivation,high transfection efficiency and no immunological rejection make it to be a good carrier of gene therapy.Endostatin is the most intensively angiogenesis inhibitor now. It can inhibit blood vessel endothelium proliferation specificly and inhibit tumor growth effectively. Especially it can inhibit powerful angiogenesis induced by tumor cells.If the carrier cells transfected by angiogenesis inhibition gene are implanted into glioma in brain, we can inhibit angiogenesis and tumor growth effectively. This is a new strategy in glioma therapy.On account of correlative research foundation in domestic and in abroad, our research applys endostatin gene and MSCs to treat glioma in rat brain. It include three parts:Part one: Isolation,culture of rat bone marrow mesenchymal stem cells and identification of multi-directional differentiation.[Objective]: To establish method of isolation and culture of rat bone marrow mesenchymal stem cells in vitro and to confirm the potency of multi-directional differentiation by cartilage cell,fat cell and nerve cell formation induction. [Method]: Primary and subcultured MSCs are cultivated in DMEM-LG with 10% fatal bovine serum(FBS) after gradient centrifugation in Peroll; MSCs, quality is evaluated with morphology and surface marker; States of MSCs growing in culture are observed with inverted microscope; MSCs are induced to cartilage cell,fat cell and nerve cell by cartilage cell,fat cell and nerve cell formation induction, and they are identifyed by alkaline phosphatase staining,oil red O staining and immunohistochemistry staining; [Result]: Primary MSCs present lymphoid form at first and total yield is achieved in 8-10 days, then the morphology of them becomes circular, polygonal or irregular forms. The cells stretch gradually after subcultured cultivation and present shuttle, spindle forms. After several generations, they are more and more like fibroblastic cells; CD29 and CD44 are expressed positively by flow cytometry; The cytoplasm of MSCs appears brownish-black by alkaline phosphatase staining after 21 days cartilage cell induction. A great quantity fatty appear in the cell of MSCs by oil red O staining after 2 weeks fat cell induction. Typical neuron cell appear after 24 hours nerve cell induction, NSE and Nestin are expressed positively by immunohistochemistry staining. [Conclusion]:Succeed in cultivation of primary and subcultured MSCs and MCSs have the potency of multi-directional differentiation.Part two: Experiment study in construction of pcDNA3-Endo eukaryon expression plasmid and angiogenesis inhibition in vitro.[Objective]:To construct the pcDNA3-Endo eukaryon expression plasmid and to establish MSCs system of pcDNA3-Endo transfection stably. [Method]: Endostatin mRNA is extracted from rat liver by Trizol and endostatin cDNA is synthetized by RT-PCR; Endostatin cDNA and pcDNA3 are connected and pcDNA3-Endo recombined plasmid is constructed successfully; pcDNA3-Endo is transfected into MSCs by Lipofectamine; The expression of endostatin is identifyed by RT-PCR and Western Blot;Endostatin proteinum activity is detected by ECV-304 cell proliferation inhibition experiment. [Result]: A strip comes to appear in the position of 680bp after gelose electrophoresis and its size accord with endostatin cDNA; Sequencing result accords with the endostatin cDNA announced by GeneBank;Two strips come to appear in the position of 5.4kb and 680bp after gelose electrophoresis and their size accord with pcDNA3 and endostatin cDNA accordingly; Two positive strips appear in the position of 680bp,20kD after RT-PCR and WesternBlot gelose electrophoresis;The growth of ECV-304 cell is inhibited obviously by endostatin after ECV-304 cell proliferation inhibition experiment. [Conclusion]: Succeed in construction of pcDNA3- Endo eukaryon expression plasmid and MSCs system of pcDNA3-Endo transfection stably.Part three: Experiment study in the treatment of glioma in rat brain by MSCs transfected by pcDNA3-Endo in vivo.[Objective]:To confirm the validity of glioma treatment in rat brain by pcDNA3-Endo combined with MSCs. [Method]:Rat glioma model is established by stereotaxis technology; Four groups are established randomly(pcDNA3-Endo-MSCs group;pcDNA3- Endo group;MSCs group;control group); The difference of treatment effect in each group are contrasted by distribution of MSCs marked by DAPI,life span,tumor volume in different time,HE staining,transmission electron microscope and necrosis ratio and blood vessel number; The expression of endostatin is detected by RT-PCR and WesternBlot. [Result]: Life span is extended obviously in pcDNA3- Endo-MSCs treatment group; Tumor volume in different time is diminished obviously in pcDNA3-Endo-MSCs treatment group; Blood vessel number is diminished obviously and necrosis increases in pcDNA3-Endo-MSCs treatment group by HE staining; The apoptosis structure appears in pcDNA3-Endo-MSCs treatment group including nucelus pycnosis,chromatin agglutination,apoptotic body appearance and so on; Necrosis ratio in pcDNA3-Endo-MSCs treatment group is obviously larger than other non-treatment group, but blood vessel number in pcDNA3-Endo-MSCs treatment group is obviously lower than other non-treatment group; Endostatin can be expressed effectively by RT-PCR and Western Blot detection. [Conclusion]:The treatment of glioma by MSCs transfected by pcDNA3-Endo is more effective than using single pcDNA3-Endo plasmid and using single MSCs, application of pcDNA3-Endo combined with MSCs is a developmental therapeutic tool.
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