發(fā)布時間：2018-03-07 20:58

  本文選題：膠原基仿生骨基質(zhì)　切入點：纖維蛋白止血敷料　出處：《四川農(nóng)業(yè)大學》2010年碩士論文　論文類型：學位論文

【摘要】： 免疫系統(tǒng)是人體內(nèi)重要的防御性系統(tǒng),免疫系統(tǒng)的任何結(jié)構(gòu)改變和功能失調(diào),將使機體免疫抗病能力降低,引起各種感染性疾病、自身免疫性疾病或腫瘤。近年來,生物藥品和生物材料新產(chǎn)品層出不窮,而由于新產(chǎn)品添加成分不當而對人體健康產(chǎn)生危害的事例也日漸增多,人們越來越關(guān)注生物藥品和生物材料的使用安全性。而對其使用安全性評價的一個重要方面即是對生物材料免疫安全性的評價。 本研究選取了兩種膠原蛋白類植入性生物材料——膠原基仿生骨基質(zhì)和纖維蛋白止血敷料,根據(jù)相關(guān)潛在免疫毒性效應(yīng)的免疫反應(yīng)的類型,針對炎癥、免疫抑制和免疫刺激3種免疫效應(yīng)進行研究,設(shè)計了大鼠體內(nèi)埋植實驗,研究這兩種生物材料是否會對大鼠免疫系統(tǒng)造成影響。主要研究內(nèi)容及結(jié)果如下： 1.選取Wistar大鼠作為實驗動物,將膠原基仿生骨基質(zhì)和羥基磷灰石進行大鼠股骨側(cè)埋植,將纖維蛋白止血敷料、纖維蛋白原和膠原蛋白進行大鼠肝臟埋植,至少暴露28d；選擇了適應(yīng)于實驗動物埋植部位的最大埋植量,即膠原基仿生骨基質(zhì)材料埋植1cm×0.1cm×0.3cm,羥基磷灰石埋植1cm×0.1cm×0.1cm,纖維蛋白止血敷料埋植1cm×1cm×0.4 cm,纖維蛋白原埋植1cm×1cm×0.4 cm,膠原蛋白埋植1cm×1cm×0.4cm。同時設(shè)免疫抑制組(即腹腔注射10mg/mL環(huán)磷酰胺)作為陽性對照,假手術(shù)組作為陰性對照,將已有的無免疫毒性或低免疫毒性的材料(羥基磷灰石、纖維蛋白原和膠原蛋白)作為平行對照。 2.對大鼠進行癥狀和體征觀察、白細胞、淋巴細胞及中性粒細胞絕對值計數(shù)、免疫器官指數(shù)分析、免疫器官組織學分析、NK細胞殺傷活力檢測、T淋巴細胞亞群分析、巨噬細胞分泌功能檢測和血清中免疫球蛋白IgG和IgM的測定,實驗結(jié)果顯示： (1)腹腔注射10mg/mL環(huán)磷酰胺(30mg/kg體重),連續(xù)4天可成功建成大鼠免疫抑制模型。免疫抑制組大鼠出現(xiàn)明顯的口鼻黏膜出血、面部腫大；免疫器官出現(xiàn)明顯病變；免疫抑制組大鼠白細胞、淋巴細胞及中性粒細胞絕對值計數(shù)、免疫器官指數(shù)、NK細胞殺傷活力、T淋巴細胞亞群、巨噬細胞分泌IL-1、IL-6、TNF-α和血清中免疫球蛋白IgG和IgM含量均顯著低于陰性對照組(P0.05)； (2)各埋植組(膠原基仿生骨基質(zhì)、纖維蛋白止血敷料、羥基磷灰石、纖維蛋白原和膠原蛋白埋植)大鼠在術(shù)后與陰性對照組相比,進食飲水正常,精神狀態(tài)良好,傷口愈合良好；體重增加正常,與陰性對照組相比無顯著差異(P0.05)； (3)各埋植組大鼠白細胞均未見異常減少和增多,白細胞計數(shù)與陰性對照組相比無顯著差異(P0.05)；手術(shù)后第2周開始,各埋植組的淋巴細胞絕對值恢復至正常水平,與陰性對照組相比無顯著差異(P0.05)； (4)各埋植組大鼠在術(shù)后與陰性對照組相比,脾臟指數(shù)和肝臟指數(shù)均無顯著差異(P0.05)； (5)免疫器官病理學切片分析結(jié)果表明,各埋植組大鼠在進行手術(shù)后與陰性對照組相比,埋植部位組織、脾臟、胸腺和腹腔淋巴結(jié)均無病變,結(jié)構(gòu)正常； (6)各埋植組大鼠在術(shù)后與陰性對照組相比,NK細胞殺傷活力無顯著性差異(p0.05),并且與NK細胞自然死亡率有顯著性差異(P0.05)； (7)各埋植組大鼠在術(shù)后與陰性對照組相比,除第2周有微弱上升外,CD3+CD4+T淋巴細胞含量、CD3+CD8+T淋巴細胞含量均無大幅變化,整體趨勢平穩(wěn),CD4+/CD8+比值也沒有出現(xiàn)下降,與陰性對照組無顯著性差異(P0.05)； (8)血清中IL-1、IL-6和TNF-α含量與陰性對照組相比血清中IL-1、IL-6和TNF-α含量各埋植組和骨假手術(shù)組無明顯差異(P0.05)； (9)各埋植組大鼠血清中免疫球蛋白IgG的含量與陰性對照組相比無顯著差異(p0.05)；大鼠血清中幾乎無IgM存在,提示未有新近感染發(fā)生。 綜合以上實驗結(jié)果,膠原基仿生骨基質(zhì)和纖維蛋白止血敷料對大鼠的免疫系統(tǒng)無明顯毒性作用,材料具有良好的免疫安全性。
[Abstract]:The immune system is an important defensive system in the human body, any structural changes and functional dysregulation of the immune system, will enable the immune resistance reduced, causing a variety of infectious diseases, autoimmune diseases or cancer. In recent years, bio pharmaceutical and biological materials and new products emerge in endlessly, and new products due to improper ingredients harm case on human health is also increasing, people pay more and more attention to the use of safety of biological pharmaceutical and biological materials. In an important aspect of the use of safety evaluation is an evaluation of the biological material of immunity.
This study selected two classes of biological material implanted collagen and collagen based biomimetic bone matrix and fibrin dressing, depending on the type of immune response, immune related potential toxic effects on inflammation, immune suppression and immune stimulating 3 immune effect was studied, the design of implant test in rats, study the two species whether the material will affect the immune system of rats. The main research contents and results are as follows:
1. Wistar rats were selected as experimental animal, collagen based biomimetic bone matrix and hydroxyapatite femur of rats implanted, the fibrin dressing, fibrinogen were implanted in rat liver and collagen, at least 28d exposure; choose to adapt to the maximum amount of implants in the experimental animal implantation sites, namely collagenic bionic bone the matrix material implanted 1cm * 0.1cm * 0.3cm, 1cm * 0.1cm * 0.1cm implantation of hydroxyapatite, fibrin dressing preparetions 1cm * 1cm * 0.4 cm, fibrinogen preparetions 1cm * 1cm * 0.4 cm * 1cm * 0.4cm. 1cm collagen implants and immunosuppression group (i.e. 10mg/mL intraperitoneal injection of cyclophosphamide) as a positive control, false operation group as negative control, material will have no immune toxicity or low immune toxicity (hydroxyapatite, fibrin and collagen) as a parallel control.
2. of the rats were observed, symptoms and signs of white blood cell, lymphocyte and neutrophil counts, analysis of immune organ index, analysis of immune organs, NK cell killing activity detection of T lymphocyte subsets, Macrophage Secretion of immunoglobulin IgG and IgM detection in the serum and the experimental results show:
(1) 10mg/mL intraperitoneal injection of cyclophosphamide (30mg/kg weight) for 4 consecutive days, can be successfully built rat model of immune suppression. The immunosuppressive rats appeared obvious nasal mucosal bleeding, facial swelling; immune organ lesions; immune suppression of white blood cells in rats, lymph cell and neutrophil counts, immune organ index, NK cell activity and T lymphocyte subsets, macrophages IL-1, IL-6, immunoglobulin IgG and IgM content in serum and TNF- alpha were significantly lower than the negative control group (P0.05);
(2) the implant group (collagen based biomimetic bone matrix, fibrin dressing, hydroxyapatite, fibrin and collagen implants) in rats after operation compared with negative control group, normal eating and drinking, a good mental state, good wound healing; normal weight, no significant difference compared with the negative control group (P0.05);
(3) the implant group rats showed no abnormal white blood cells decreased and increased, no significant differences in white blood cell count and negative control group (P0.05); second weeks after operation, the implanted group the absolute value of lymphocyte returned to normal levels, no significant difference compared with the negative control group (P0.05);
(4) there was no significant difference between the spleen index and the liver index (P0.05) compared with the negative control group.
(5) the pathological sections of immune organs showed that there was no pathological changes in the tissues, spleen, thymus and abdominal lymph nodes in all implanted rats after operation.
(6) there was no significant difference in the killing activity of NK cells between the implanted group and the negative control group after operation (P0.05), and there was a significant difference in the natural death rate between the NK cells and the control group (P0.05).
(7) the implant group rats after operation compared with negative control group, in addition to the second week weak rising, CD3+CD4+T lymphocyte content, CD3+CD8+T lymphocyte content had no significant change in the overall trend of steady, CD4+/CD8+ ratio did not decline, and the negative control group no significant difference (P0.05);
(8) the content of serum IL-1, IL-6 and TNF- alpha in the serum was not significantly different from that in the negative control group, and there was no significant difference in the content of IL-1, IL-6 and TNF- alpha in the implant group and the bone sham operation group (P0.05).
(9) there was no significant difference in serum immunoglobulin IgG between the implanted group and the negative control group (P0.05). There was almost no IgM in the serum of rats, suggesting no new infection occurred.
Based on the above results, collagen based biomimetic bone matrix and fibrin hemostatic dressing have no obvious toxic effects on the immune system in rats, and the materials have good immune safety.

【學位授予單位】：四川農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392.9

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