放射性泪道探针制备及抑制泪道再狭窄的作用与机制研究
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摘要
泪道堵塞是眼科常见病,不及时治疗病人将终生流泪,一些患者则发展为慢性泪囊炎,严重者可造成化脓性角膜炎、角膜穿孔、眼内炎,甚至失明严重影响患者的生活质量。目前临床治疗方法很多,但因治疗后出现瘢痕收缩再狭窄而影响疗效,本研究通过发明和制作放射性核素泪道探针,用其低能量的γ射线辐射泪道,抑制其结缔组织及纤维组织增生使排泪通畅,以达到治疗泪道堵塞的目的。本研究用2.0-2.5kg 左右新西兰大耳白兔制作泪道狭窄模型,对狭窄段用125I 粒子进行辐射治疗,并进行影像学、组织学和分子生物学观察。结果表明放射性125I 辐射治疗后,有明显的治疗作用和抑制再狭窄的作用,其作用机制是创伤早期小剂量的γ射线照射可抑制上皮细胞及结缔组织细胞增生和引起上皮细胞和基底膜下的结缔组织细胞凋亡,抑制病理性增生,从而防止泪道再狭窄。在泪道的治疗中探通术早期应用放射性核素近距离辐射治疗是预防再狭窄提高近远期疗效,安全、价廉的有效手段。
Objective
Stenosis of lacrimal passages is one of the common diseases in northeast area of China, which has an especially high incidence in senior individuals. At present, practicing probing of lacrimal passage is a current strategy for treating this disease. However, most patients suffer restenosis after the treatment in a few months, which decreases this strategy's short-and long-term effects far from expectations. Thus, seeking for a rapid and effective proposal, with higher security and less trauma, for preventing restenosis of lacrimal passages, is a formidable task for the time being. In this project, we practiced brachytherapy with I25I seeds imbedded in rabbit lacrimal passages to prevent restenosis by internal irradiation to special regions, where the hyperplasia tissues after probing of lacrimal passages compose of smooth muscles, fibrous tissues, blood vessels, glad epithelium and other components. Irradiation energy can induce cell apoptosis by affecting every phase and stage of cell cycles. Our purpose is
Results 1. Researches on lacrimal passage radioactive probe and related security tests We prepared lacrimal passage radioactive probe with MSI-125 I25I seeds and thermophlic plastic tubes. Further phantom tests found that, absorption dose rates between different groups change significantly among 0-13mm to the point radioactive source, while no remarkable changes can be observed in the range 13-50mm.More over, absorption dose rates in different parts of human body are under the governmental standards by probing irradiation on body surface with professional dose evaluator. 2. Experimental researches of lacrimal passage radioactive probe's inhibitory effects on preventing restenosis of post-probing of lacrimal passages We prepared stenosis mode of rabbit lacrimal passages before practicing solo probing treatment in the left passage, while at the same time, practicing probing treatment with brachytherapy of 125I seeds for internal radiation in the right passage. 7d, 30d and 90d after the imbedding, specimens were taken, 10 for each group. For all specimens, collagen V was tested through immunohistochemical staining before calculating integral optical density via computer image analysis system. Then, we compared effects of the two-treatments for inhibiting restenosis of lacrimal passages. Immunohistochemical staining demonstrated that expression of collagen V in the group treated with I25I seeds for internal radiation is significantly lower than that in the group solo adopted probing treatment, with remarkable IOD changes(pO.Ol). 3. Mechanism researches of lacrimal passage radioactive probe's inhibitory effects on preventing restenosis of post-probing of lacrimal
passages Before getting specimens, we practiced internal radiation to rabbit lacrimal passage restenosis modes 1 d, 4d, 7d, 15d, respectively. We expected to explain the molecular mechanism through comparing the tests results of irradiation group with normal group and control group, which are lacrimal passage restenosis modes without irradiation. The expressions of protein bcl-2, bax, TGF-Pi, bFGF in lacrimal passages were qualitatively determined through immunohistochemistry method and semi-quantitatively determined through Western blot. And, mRNA expression levels of such genes in these groups were detected through in situ hybridization. The results showed that, protein bcl-2 expression in control group is higher than that in normal group, while the expression in irradiation group is significantly lower than that in control group. The results demonstrated corresponding changes between bcl-2 mRNA and protein. However, bax protein expression in epithelium of normal group is higher than that in control group. Expressions of protein bax in stromal cells between normal group and control group show no significance.Meamwhile, bcl-2 protein expression in epithelium and stroma of control group is higher than bax. While after internal radiation, protein bax in epithelium is remarkably higher than that in control group. For bax mRNA expression, it is higher in normal epithelium than that in control group. Compared with control group, after y radiation, expressions of bax mRNA in epithelium and stroma are greatly enhanced. Compared control group with normal group, expressions of TGF-(i| protein in epithelium and stroma show no significances. After irradiation, TGF-Pi protein express increasingly in epithelium and stroma of lacrimal passages. For TGF-Pi mRNA expressions, no significance was found between normal group and control group. However, after irradiation, TGF-Pi positive
rate increased in epithelium and stroma cells, which showed great significance compared with control group. While for bFGF protein expressions, it is higher in control group than that in normal group. However, after internal irradiation, bFGF protein decreased in epithelium and stroma. The bFGF mRNA expression demonstrated corresponding change, namely higher than normal group. While the bFGF mRNA positive rate decreased. We analyzed cell apoptosis after y radiation in lacrimal passage through TUNEL staining, flow-cytometry and DNA agarose gel electrophoresis. Quite a few cell apoptosis appears in normal group and scanty cell apoptosis can be observed in control group. The results show no significance between these two groups. However, after adopted y irradiation, cell apoptosis was accelerated remarkably, which is increased as time accumulates. We can find an apoptosis peak before the diploid peak. The apoptosis peak reaches maximum 7d after the irradiation, when the genome shows typical apoptosis change, namely DNA Ladder. Conclusion Though this project, we found that hyperplasia of collagen V has been reduced after y irradiation. Irradiation dose tests shows that practicing this strategy is safe to both patients and operators according to results of surface , dose detection of human body. The y irradiation can affect lacrimal passage cells hyperplasia and induce apoptosis both in vivo and in vitro, through results from multiple detection. The mechanism for the effects is related to changes of expressions of bcl-2, bax, TGF-Pi and bFGF mRNAs and proteins. This experimental research focused on improving clinical effects of probing treatment to lacrimal passages, and provided important theoretical significance
Objective
Stenosis of lacrimal passages is one of the common diseases in northeast area of China, which has an especially high incidence in senior individuals. At present, practicing probing of lacrimal passage is a current strategy for treating this disease. However, most patients suffer restenosis after the treatment in a few months, which decreases this strategy's short-and long-term effects far from expectations. Thus, seeking for a rapid and effective proposal, with higher security and less trauma, for preventing restenosis of lacrimal passages, is a formidable task for the time being. In this project, we practiced brachytherapy with I25I seeds imbedded in rabbit lacrimal passages to prevent restenosis by internal irradiation to special regions, where the hyperplasia tissues after probing of lacrimal passages compose of smooth muscles, fibrous tissues, blood vessels, glad epithelium and other components. Irradiation energy can induce cell apoptosis by affecting every phase and stage of cell cycles. Our purpose is
Results 1. Researches on lacrimal passage radioactive probe and related security tests We prepared lacrimal passage radioactive probe with MSI-125 I25I seeds and thermophlic plastic tubes. Further phantom tests found that, absorption dose rates between different groups change significantly among 0-13mm to the point radioactive source, while no remarkable changes can be observed in the range 13-50mm.More over, absorption dose rates in different parts of human body are under the governmental standards by probing irradiation on body surface with professional dose evaluator. 2. Experimental researches of lacrimal passage radioactive probe's inhibitory effects on preventing restenosis of post-probing of lacrimal passages We prepared stenosis mode of rabbit lacrimal passages before practicing solo probing treatment in the left passage, while at the same time, practicing probing treatment with brachytherapy of 125I seeds for internal radiation in the right passage. 7d, 30d and 90d after the imbedding, specimens were taken, 10 for each group. For all specimens, collagen V was tested through immunohistochemical staining before calculating integral optical density via computer image analysis system. Then, we compared effects of the two-treatments for inhibiting restenosis of lacrimal passages. Immunohistochemical staining demonstrated that expression of collagen V in the group treated with I25I seeds for internal radiation is significantly lower than that in the group solo adopted probing treatment, with remarkable IOD changes(pO.Ol). 3. Mechanism researches of lacrimal passage radioactive probe's inhibitory effects on preventing restenosis of post-probing of lacrimal
passages Before getting specimens, we practiced internal radiation to rabbit lacrimal passage restenosis modes 1 d, 4d, 7d, 15d, respectively. We expected to explain the molecular mechanism through comparing the tests results of irradiation group with normal group and control group, which are lacrimal passage restenosis modes without irradiation. The expressions of protein bcl-2, bax, TGF-Pi, bFGF in lacrimal passages were qualitatively determined through immunohistochemistry method and semi-quantitatively determined through Western blot. And, mRNA expression levels of such genes in these groups were detected through in situ hybridization. The results showed that, protein bcl-2 expression in control group is higher than that in normal group, while the expression in irradiation group is significantly lower than that in control group. The results demonstrated corresponding changes between bcl-2 mRNA and protein. However, bax protein expression in epithelium of normal group is higher than that in control group. Expressions of protein bax in stromal cells between normal group and control group show no significance.Meamwhile, bcl-2 protein expression in epithelium and stroma of control group is higher than bax. While after internal radiation, protein bax in epithelium is remarkably higher than that in control group. For bax mRNA expression, it is higher in normal epithelium than that in control group. Compared with control group, after y radiation, expressions of bax mRNA in epithelium and stroma are greatly enhanced. Compared control group with normal group, expressions of TGF-(i| protein in epithelium and stroma show no significances. After irradiation, TGF-Pi protein express increasingly in epithelium and stroma of lacrimal passages. For TGF-Pi mRNA expressions, no significance was found between normal group and control group. However, after irradiation, TGF-Pi positive
rate increased in epithelium and stroma cells, which showed great significance compared with control group. While for bFGF protein expressions, it is higher in control group than that in normal group. However, after internal irradiation, bFGF protein decreased in epithelium and stroma. The bFGF mRNA expression demonstrated corresponding change, namely higher than normal group. While the bFGF mRNA positive rate decreased. We analyzed cell apoptosis after y radiation in lacrimal passage through TUNEL staining, flow-cytometry and DNA agarose gel electrophoresis. Quite a few cell apoptosis appears in normal group and scanty cell apoptosis can be observed in control group. The results show no significance between these two groups. However, after adopted y irradiation, cell apoptosis was accelerated remarkably, which is increased as time accumulates. We can find an apoptosis peak before the diploid peak. The apoptosis peak reaches maximum 7d after the irradiation, when the genome shows typical apoptosis change, namely DNA Ladder. Conclusion Though this project, we found that hyperplasia of collagen V has been reduced after y irradiation. Irradiation dose tests shows that practicing this strategy is safe to both patients and operators according to results of surface , dose detection of human body. The y irradiation can affect lacrimal passage cells hyperplasia and induce apoptosis both in vivo and in vitro, through results from multiple detection. The mechanism for the effects is related to changes of expressions of bcl-2, bax, TGF-Pi and bFGF mRNAs and proteins. This experimental research focused on improving clinical effects of probing treatment to lacrimal passages, and provided important theoretical significance
引文
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