胶原基仿生骨基质和纤维蛋白止血敷料的免疫安全性研究

发布时间：2018-03-07 20:58

本文选题：胶原基仿生骨基质　切入点：纤维蛋白止血敷料　出处：《四川农业大学》2010年硕士论文　论文类型：学位论文

【摘要】： 免疫系统是人体内重要的防御性系统,免疫系统的任何结构改变和功能失调,将使机体免疫抗病能力降低,引起各种感染性疾病、自身免疫性疾病或肿瘤。近年来,生物药品和生物材料新产品层出不穷,而由于新产品添加成分不当而对人体健康产生危害的事例也日渐增多,人们越来越关注生物药品和生物材料的使用安全性。而对其使用安全性评价的一个重要方面即是对生物材料免疫安全性的评价。本研究选取了两种胶原蛋白类植入性生物材料——胶原基仿生骨基质和纤维蛋白止血敷料,根据相关潜在免疫毒性效应的免疫反应的类型,针对炎症、免疫抑制和免疫刺激3种免疫效应进行研究,设计了大鼠体内埋植实验,研究这两种生物材料是否会对大鼠免疫系统造成影响。主要研究内容及结果如下： 1.选取Wistar大鼠作为实验动物,将胶原基仿生骨基质和羟基磷灰石进行大鼠股骨侧埋植,将纤维蛋白止血敷料、纤维蛋白原和胶原蛋白进行大鼠肝脏埋植,至少暴露28d；选择了适应于实验动物埋植部位的最大埋植量,即胶原基仿生骨基质材料埋植1cm×0.1cm×0.3cm,羟基磷灰石埋植1cm×0.1cm×0.1cm,纤维蛋白止血敷料埋植1cm×1cm×0.4 cm,纤维蛋白原埋植1cm×1cm×0.4 cm,胶原蛋白埋植1cm×1cm×0.4cm。同时设免疫抑制组(即腹腔注射10mg/mL环磷酰胺)作为阳性对照,假手术组作为阴性对照,将已有的无免疫毒性或低免疫毒性的材料(羟基磷灰石、纤维蛋白原和胶原蛋白)作为平行对照。 2.对大鼠进行症状和体征观察、白细胞、淋巴细胞及中性粒细胞绝对值计数、免疫器官指数分析、免疫器官组织学分析、NK细胞杀伤活力检测、T淋巴细胞亚群分析、巨噬细胞分泌功能检测和血清中免疫球蛋白IgG和IgM的测定,实验结果显示： (1)腹腔注射10mg/mL环磷酰胺(30mg/kg体重),连续4天可成功建成大鼠免疫抑制模型。免疫抑制组大鼠出现明显的口鼻黏膜出血、面部肿大；免疫器官出现明显病变；免疫抑制组大鼠白细胞、淋巴细胞及中性粒细胞绝对值计数、免疫器官指数、NK细胞杀伤活力、T淋巴细胞亚群、巨噬细胞分泌IL-1、IL-6、TNF-α和血清中免疫球蛋白IgG和IgM含量均显著低于阴性对照组(P0.05)； (2)各埋植组(胶原基仿生骨基质、纤维蛋白止血敷料、羟基磷灰石、纤维蛋白原和胶原蛋白埋植)大鼠在术后与阴性对照组相比,进食饮水正常,精神状态良好,伤口愈合良好；体重增加正常,与阴性对照组相比无显著差异(P0.05)； (3)各埋植组大鼠白细胞均未见异常减少和增多,白细胞计数与阴性对照组相比无显著差异(P0.05)；手术后第2周开始,各埋植组的淋巴细胞绝对值恢复至正常水平,与阴性对照组相比无显著差异(P0.05)； (4)各埋植组大鼠在术后与阴性对照组相比,脾脏指数和肝脏指数均无显著差异(P0.05)； (5)免疫器官病理学切片分析结果表明,各埋植组大鼠在进行手术后与阴性对照组相比,埋植部位组织、脾脏、胸腺和腹腔淋巴结均无病变,结构正常； (6)各埋植组大鼠在术后与阴性对照组相比,NK细胞杀伤活力无显著性差异(p0.05),并且与NK细胞自然死亡率有显著性差异(P0.05)； (7)各埋植组大鼠在术后与阴性对照组相比,除第2周有微弱上升外,CD3+CD4+T淋巴细胞含量、CD3+CD8+T淋巴细胞含量均无大幅变化,整体趋势平稳,CD4+/CD8+比值也没有出现下降,与阴性对照组无显著性差异(P0.05)； (8)血清中IL-1、IL-6和TNF-α含量与阴性对照组相比血清中IL-1、IL-6和TNF-α含量各埋植组和骨假手术组无明显差异(P0.05)； (9)各埋植组大鼠血清中免疫球蛋白IgG的含量与阴性对照组相比无显著差异(p0.05)；大鼠血清中几乎无IgM存在,提示未有新近感染发生。综合以上实验结果,胶原基仿生骨基质和纤维蛋白止血敷料对大鼠的免疫系统无明显毒性作用,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.

【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.9

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