本文選題：刺槐素 + 血腦屏障　； 參考：《廈門大學》2014年碩士論文

【摘要】：腦膠質瘤為起源于神經膠質細胞的腫瘤,是最常見的原發(fā)性顱內腫瘤。尤其是多形性腦膠質瘤(GBM),它是一類較頻繁發(fā)生的中樞神經系統(tǒng)性惡性腫瘤,較容易擴散到周圍的正常組織。傳統(tǒng)的手術治療很難將腦膠質瘤全部切除,而且很容易復發(fā)。目前,最有效的治療腦膠質瘤的方法是外科手術結合化療,但是化療治療效果非常有限,還會造成系統(tǒng)的許多副作用。這些副作用主要是由于存在血腦屏障(BBB),不允許98%的小分子和100%的大分子通過,化療藥物很難通過BBB,而積累在周圍組織產生副作用。因此我們應當設計一種藥物載體來傳遞化療藥物,使其通過血腦屏障并靶向于腫瘤病灶部位。低密度脂蛋白受體相關蛋白(LRP)是存在于BBB上的一種受體,IgG被LRP識別,并通過LRP調節(jié)的內吞途徑通過血腦屏障。選擇刺槐素作為模型藥物。刺槐素難溶于水,近幾年越來越多的研究表明刺槐素具有保護神經和抗腫瘤的作用。因此我們以脂質體作為載體來傳遞刺槐素到腦膠質瘤所在部位。以IgG作為靶向材料,引導脂質體通過BBB并靶向于腦膠質瘤U87 MG細胞。這一新型的脂質體可以被用來作為抗腦膠質瘤藥物的理想劑型,同時也可作為其他中樞神經系統(tǒng)疾病治療藥物的傳遞載體。本文研究的主要內容包括以下幾個方面:1.合成了 CL-PEG2000-Mal,活化的IgG蛋白與其結合,制備的主動靶向脂質體通過LRP調節(jié)的細胞內吞途徑進入細胞。2.建立了刺槐素的體外高效液相分析方法和刺槐素脂質體包封率的測定方法(微孔濾膜過濾分離-HPLC檢測藥物含量),薄膜分散法制備脂質體,擠出、微孔濾膜過濾達到分離游離藥物和整粒的目的。刺槐素脂質體的最佳處方為磷脂的含量5%(w/v),總磷脂與膽固醇之比為4:1,藥物與磷脂之比為1:20,水化介質為10%海藻糖。IgG修飾的刺槐素脂質體的包封率大于80%,平均粒徑為163 nm,PDI為0.352,IgG修飾的脂質體的形狀為規(guī)則的類球形,擁有良好的體外釋放行為。3.MTT實驗和細胞攝取實驗證明了 IgG修飾的空白脂質體低毒而且能夠明顯增加U87 MG細胞和bEnd.3細胞對脂質體的攝取。在MTT實驗中,與普通刺槐素脂質體和游離刺槐素相比,IgG修飾的刺槐素脂質體對U87 MG細胞增殖的抑制作用最強。在細胞的攝取實驗中,U87MG細胞對IgG修飾的脂質體的攝取指數分別是對照組1.22、1.91、2.20、2.27、2.55倍,bEnd.3細胞對脂質體的攝取效果與U87 MG細胞相似。4.轉移通過血腦屏障能力實驗和體外雙靶向性實驗都表明了 IgG修飾的脂質體可以高效的通過BBB,靶向于U87MG細胞,而且通過BBB后的脂質體維持著完整性�？傊�,在本研究的體外的共培養(yǎng)模式中,IgG修飾的脂質體不僅增加了其通過BBB的能力還能夠連續(xù)靶向于U87 MG細胞�？傊�,IgG修飾的刺槐素脂質體的MTT實驗證實了 IgG球蛋白修飾提高了刺槐素的細胞毒性,U87 MG細胞和bEnd.3細胞的攝取實驗、抑制性試驗都證明IgG的修飾提高了脂質體的攝取量,而且具有一定的靶向性。脂質體的透過能力試驗和體外雙靶向性實驗與預期實驗結果相同,達到既能夠通過血腦屏障又能夠靶向U87MG細胞,起到連續(xù)靶向性的效果。IgG的修飾提高了脂質體的腦靶向性,具有良好的臨床應用前景。
[Abstract]:Glioma, the most common primary intracranial tumor, is the most common primary intracranial tumor, especially the GBM. It is a kind of more frequent central nervous system malignant tumor, which is easy to spread to normal tissues around. It is difficult to remove all the glioma by traditional surgical treatment, and it is very easy. Recrudesce. Currently, the most effective treatment of glioma is surgery combined with chemotherapy, but the effect of chemotherapy is very limited and can cause many side effects of the system. These side effects are mainly due to the existence of the blood brain barrier (BBB), which does not allow 98% small molecules and 100% large molecules to pass through, and the chemotherapeutic drugs are difficult to accumulate through BBB. The surrounding tissue produces side effects. Therefore, we should design a drug carrier to pass the chemotherapeutic agents through the blood brain barrier and target the site of the tumor. Low density lipoprotein receptor related protein (LRP) is a receptor on BBB, IgG is identified by LRP, and through the LRP regulated endocytic pathway through the blood brain barrier. Select spines. Sophotin is a model drug. Robinia is difficult to dissolve in water. In recent years, more and more studies have shown that Robinia has a protective effect on nerve and antitumor. Therefore, we use liposome as a carrier to transfer Robinia to the location of brain glioma. IgG is used as a target material to guide liposomes through BBB and target glioma U87 MG cells. This new liposome can be used as an ideal form of anti glioma drugs, and can also be used as a carrier for other central nervous system diseases. The main contents of this study include the following aspects: 1. synthesis of active IgG proteins and the active targeting liposomes are synthesized by CL-PEG2000-Mal. The high performance liquid phase analysis method of Robinia in vitro and the method for determining the encapsulation efficiency of Robinia Robinia liposome were established by the LRP regulated endocytic pathway into the cell.2., and the liposomes were prepared by the microporous filter membrane filtration, and the liposomes were prepared by the thin film dispersion method. The purpose of separating free drugs and whole particles was achieved by extrusion and microporous filtration membrane filtration. The best prescription of sophiotin liposome is 5% (w/v), the ratio of total phospholipid to cholesterol is 4:1, the ratio of drug to phospholipid is 1:20. The encapsulation efficiency of Sophora japonica liposomes modified by 10% trehalose.IgG is more than 80%, the average particle size is 163 nm, PDI is 0.352, the shape of IgG trimming liposome is a regular spherical shape, and has good shape. In vitro release behavior.3.MTT and cell uptake experiments demonstrate that IgG modified blank liposomes are low toxic and can significantly increase the uptake of liposomes by U87 MG cells and bEnd.3 cells. In MTT experiments, the inhibition of IgG modified Robinia liposomes on the proliferation of U87 MG cells in comparison with the normal Robinia liposomes and free Robinia In the cell uptake experiments, the uptake of IgG modified liposomes by U87MG cells was 1.22,1.91,2.20,2.27,2.55 times of the control group. The effect of bEnd.3 cells on the liposomes was similar to that of U87 MG cells,.4. transfer through the blood brain barrier ability experiment and in vitro double targeting experiment all showed IgG modified liposomes. It is possible to efficiently target U87MG cells through BBB, and maintain integrity through the liposomes after BBB. In a word, in the co culture model of this study, IgG modified liposomes not only increase their ability to pass through BBB but also target U87 MG cells continuously. In a word, the MTT experiment of the IgG modified Robinia liposomes confirms I. GG globulin modification improves the cytotoxicity of Robinia, the uptake of U87 MG cells and bEnd.3 cells, and the inhibition test shows that the modification of IgG improves the uptake of liposomes and has a certain target. The permeability test of liposomes and the double targeting experiment in vitro are the same as the expected experimental results. The blood-brain barrier can also target U87MG cells and play a continuous targeting role. The modification of.IgG improves the targeting ability of liposomes and has good clinical application prospects.
【學位授予單位】：廈門大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R943

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