【摘要】：目的:應用博來霉素(bleomycin,BLM)誘導大鼠構建肺纖維化模型,探討基質金屬蛋白酶-9(matrix metalloproteinase-9,MMP-9)、基質金屬蛋白酶抑制劑-1(tissue inhibitor of metalloproteinase-1,TIMP-1)在肺纖維化過程中的表達及應用外源性硫化氫的干預作用。方法:健康雄性SD大鼠共45只,按隨機數字表法分為3組(每組15只),即對照組、BLM組、BLM+Na HS(硫化氫供體)組。BLM組、BLM+Na HS組按5mg/kg劑量給予BLM溶液一次性氣管內注入建立肺纖維化模型,對照組氣管內注入等體積的生理鹽水,造模后第一天開始,BLM+Na HS組每天腹腔注射Na HS溶液(28μmol/kg);BLM組和對照組每天腹腔注射等體積生理鹽水。造模后分別在第7,14,28天進行取材,各組每次隨機處死5只大鼠。取左肺組織放于4%甲醛固定后做HE染色和Masson染色電鏡觀察肺泡炎和肺纖維化改變,免疫組織化學染色法檢測肺組織中MMP-9、TIMP-1蛋白的表達情況;取右肺組織進行RT-PCR檢測肺組織中MMP-9、TIMP-1 m RNA的表達情況。結果:1.HE和Masson染色結果顯示:對照組各時間段均未見炎癥及肺纖維化改變,BLM組7d以炎癥反應為主,14d開始出現纖維區(qū),肺泡腔開始出現較多肺成纖維細胞;28d肺泡炎癥明顯減輕,肺泡腔內可見大量肌成纖維細胞及膠原沉積,肺纖維化模型形成;BLM+Na HS組也出現了肺泡炎和纖維化改變,但較BLM組有明顯減輕。2.免疫組化和RT-PCR結果顯示:對照組各時期MMP-9、TIMP-1僅有少量表達,而BLM組和BLM+Na HS組MMP-9、TIMP-1表達與對照組比較有顯著增加,差異有統(tǒng)計學意義(P㩳0.05),BLM+Na HS處理組MMP-9的表達在各時間階段較BLM組有不同程度減低,7d時MMP-9表達降低與BLM組比較差異有顯著統(tǒng)計學意義(P㩳0.01),14、28d較BLM組比較差異有統(tǒng)計學意義(P㩳0.05),BLM+Na HS組TIMP-1的表達在各時間階段較BLM組有不同程度的降低,7d時差異有統(tǒng)計學意義(P㩳0.05),14、28d時差異有顯著統(tǒng)計學意義(P㩳0.01)。3.MMP-9/TIMP-1平衡比值結果顯示:與對照組比較,BLM組MMP-9/TIMP-1平衡比值在7d時較對照組升高,差異有顯著統(tǒng)計學意義(P㩳0.01),在14、28d時比值較對照組降低,差異有統(tǒng)計學意義(P㩳0.05);BLM+Na HS組MMP-9/TIMP-1比值在7d時較BLM組下降,差異有統(tǒng)計學意義(P㩳0.05),14、28d較BLM組有升高,差異有統(tǒng)計學意義(P㩳0.05);較對照組比較,BLM+Na HS組MMP-9/TIMP-1比值在7d時有升高,差異有統(tǒng)計學意義(P㩳0.05),14、28d時比值有降低,差異無統(tǒng)計學意義。結論:1.外源性硫化氫能夠減輕博來霉素所致大鼠肺泡炎和肺纖維化的程度,具有抗肺纖維化作用。2.外源性硫化氫抗肺纖維化機制之一可能是抑制肺組織中MMP-9、TIMP-1的表達,并在一定程度上調整MMP-9和TIMP-1的比值使其趨于平衡。
[Abstract]:Aim: to investigate the expression of matrix metalloproteinase-9 (matrix metalloproteinase-9) and matrix metalloproteinase-1 (TIMP-1) in pulmonary fibrosis induced by bleomycin (BLM) and the effects of exogenous hydrogen sulfide (H2S) on the expression of matrix metalloproteinase-1 (TIMP-1) in pulmonary fibrosis. Methods: there were 45 healthy male SD rats. According to the random number table method, the rats were divided into three groups (15 rats in each group), namely, the control group was treated with BLM Na HS (hydrogen sulfide donor. The model of pulmonary fibrosis was established by intratracheal instillation of BLM solution in the BLM Na HS group according to the dose of 5mg/kg. The same volume of saline was injected into the trachea of the control group. The same volume of saline was injected intraperitoneally every day in the BLM Na HS group (28 渭 mol/kg) and the control group on the first day after the establishment of the model. After modeling, 5 rats were randomly killed in each group on day 14, 28, respectively. The changes of alveolitis and pulmonary fibrosis were observed by HE staining and Masson staining after the left lung tissue was fixed with 4% formaldehyde. The expression of MMP-9 and TIMP-1 protein was detected by immunohistochemical staining. The expression of MMP-9 and TIMP-1 m RNA was detected by RT-PCR in right lung tissue. Results 1. The results of HE and Masson staining showed that there were no inflammatory and pulmonary fibrosis changes in the control group. In the BLM group, the inflammatory reaction mainly appeared on the 14th day, and the alveolar inflammation began to appear in the alveolar cavity. The alveolar inflammation in the alveolar cavity began to appear more than 28 days, and the alveolar inflammation was obviously alleviated. A large number of myofibroblasts and collagen deposition could be seen in the alveolar cavity, and alveolitis and fibrosis were also found in the model of pulmonary fibrosis in the BLM Na HS group, but the changes of alveolitis and fibrosis were significantly reduced compared with those in the BLM group. The results of immunohistochemistry and RT-PCR showed that there was only a little expression of MMP-9 and TIMP-1 in the control group, while the expression of MMP-9 and TIMP-1 in the BLM group and BLM Na HS group was significantly higher than that in the control group. The expression of MMP-9 in BLM-Na HS group was significantly lower than that in BLM group at 7 days after treatment with BLM Na HS. There was significant difference between BLM group and BLM group (P0. 01) 1428 days after treatment, there was significant difference between BLM group and BLM group. (P0. 05) the expression of TIMP-1 in BLMNa HS group was significantly lower than that in BLM group at day 7 (P0. 05). (P0. 01). The results of MMP-9 / TIMP-1 balance ratio showed that: compared with the control group, the MMP-9/TIMP-1 balance ratio of BLM group was significantly lower than that of control group (P0. 05). On the 7th day, it was higher than that of the control group. The difference was statistically significant (P0. 01). The ratio of MMP-9/TIMP-1 in BLM Na HS group was significantly lower than that in BLM group at day 14 and 28. The ratio of MMP-9/TIMP-1 in BLM Na HS group was significantly lower than that in BLM group at day 7, and the difference was statistically significant (P0. 05) at 1428 days, which was higher than that in BLM group. Compared with the control group, the ratio of MMP-9/TIMP-1 increased at the 7th day, and decreased at 14 ~ 28 days, but there was no significant difference in the ratio between the two groups (P < 0. 05, P < 0. 05), but no significant difference was found between the control group and the control group (P < 0. 05, P < 0. 05). Conclusion 1. Exogenous hydrogen sulfide can reduce the degree of alveolitis and pulmonary fibrosis induced by bleomycin in rats. One of the mechanisms of exogenous hydrogen sulfide against pulmonary fibrosis may be to inhibit the expression of MMP-9 and TIMP-1 in lung tissues and to adjust the ratio of MMP-9 and TIMP-1 to a certain extent to balance them.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R563

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