富氢盐水对大鼠干眼模型眼表的保护作用

发布时间：2018-05-05 21:30

本文选题：大鼠干眼 + 富氢盐水　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的干眼(dry eye,DE)是眼表常见疾病之一,主要表现为眼部不适,视觉质量下降,伴有泪液渗透压升高和眼表炎症。炎症、性激素分泌失衡、神经机能障碍、细胞凋亡及氧化应激共同参与干眼的发病机制。目前,人工泪液治疗干眼虽有一定疗效,但停药后易复发。局部应用抗炎药物可抑制炎症因子,减轻干眼症状,但长期使用存在毒副作用。如何有效的控制甚至逆转干眼,寻找一种安全有效的药物迫在眉睫。研究发现,氢气具选择性抗氧化作用,同时它可抑制炎性因子的释放,具有抗炎作用。富氢盐水(hydrogen-rich saline,HRS)是将氢气在0.4MPa高压下溶于生理盐水,达到饱和浓度为0.6mmol/L,它在多种疾病的动物模型中均表现出明显抗炎作用。本实验使用HRS滴眼及腹腔注射两种途径干预东莨菪碱诱导的大鼠干眼模型,检测各组大鼠的临床指标、角结膜组织病理变化、细胞凋亡情况、鳞状上皮化生程度以及炎症相关因子核因子-kB(nuclear factor-kB,NF-kB)的表达,探讨HRS对大鼠干眼眼表是否具有保护作用。方法1.采用随机数字表法将30只6周龄健康Wistar大鼠分为6组,分别为正常组(A)、干眼组(B)、HRS滴眼组(C)、生理盐水(normal saline,NS)滴眼组(D)、HRS腹腔注射组(E)、NS腹腔注射组(F),每组5只(10眼)。后五组后肢皮下注射3mg·mL-1氢溴酸东莨菪碱建立干眼模型,每天注射4次(9点、12点、15点、18点),每次0.5ml,左右两侧交替进行。对C组和D组分别用HRS、NS点眼,每小时点眼1次,每天点眼9次(8点半-16点半);E组和F组按5ml·kg-1每天分别腹腔注射HRS、NS 1次(18点半),连续28天。2.用药前及用药后7、14、21、28天分别对各组大鼠行泪液分泌试验(SchirmerⅠtest,SⅠt)、泪膜破裂时间(break-up time,BUT)和角膜上皮荧光素钠染色评分,记录数据。3.用药后28天,颈椎脱臼法处死大鼠,取整个眼球,石蜡切片后行HE染色,光镜下观察各组大鼠角结膜组织学变化。4.行过碘酸-雪夫(periodic acid schiff stain,PAS)染色,光镜下观察各组大鼠结膜杯状上皮细胞形态,并计数光镜下每高倍视野杯状细胞数目。5.原位末端转移酶标记(TUNEL)法,荧光显微镜下检测各组大鼠角结膜凋亡情况,并计数荧光显微镜下每高倍视野细胞凋亡个数。6.行免疫组化染色,光镜下观察各组大鼠角结膜组织中角蛋白10(keratin 10,K10)的表达,评估鳞状上皮化生程度。7.取各组大鼠角结膜组织,采用免疫印记法检测各组大鼠角结膜组织NF-kB的表达。结果1.SⅠt:比较各组大鼠造模7天的SⅠt差异具有统计学意义(F=2.897,P0.05),干眼组SIt值与正常组相比下降(P0.05)。比较各组大鼠造模14天SⅠt值差异具有统计学意义(F=5.194,P0.05),HRS滴眼组和HRS腹腔注射组SⅠt值均比干眼组升高(P0.05);HRS滴眼组和HRS腹腔注射组SⅠt值分别比NS滴眼组和NS腹腔注射组升高(P0.05);且HRS滴眼组和HRS腹腔注射组SⅠt值差异始终无统计学意义(P0.05)。至28天始终保持稳定。2.BUT:比较各组大鼠造模7天BUT差异具有统计学意义(F=2.910,P0.05),干眼组BUT值与正常组相比下降(P0.05)。比较各组大鼠造模14天BUT差异具有统计学意义(F=3.894,P0.05),HRS滴眼组和HRS腹腔注射组BUT值均比干眼组升高(P0.05);HRS滴眼组和HRS腹腔注射组BUT值分别比NS滴眼组和NS腹腔注射组升高(P0.05);且HRS滴眼组和HRS腹腔注射组BUT值差异始终无统计学意义(P0.05)。至28天始终保持稳定。3.角膜上皮荧光素钠染色评分:比较各组大鼠造模7天的角膜上皮荧光素钠染色评分差异具有统计学意义(F=2.424,P0.05),干眼组染色评分与正常组相比升高(P0.05)。比较各组大鼠造模14天的角膜上皮荧光素钠染色评分差异具有统计学意义(F=16.487,P0.05),HRS滴眼组和HRS腹腔注射组染色评分均比干眼组下降(P0.05);HRS滴眼组和HRS腹腔注射组染色评分分别比NS滴眼组和NS腹腔注射组下降(P0.05);且HRS滴眼组和HRS腹腔注射组染色评分差异始终无统计学意义(P0.05)。至28天始终保持稳定。4.HE染色:光镜下观察,干眼组角膜上皮细胞分层模糊,排列不紧密,细胞体积较大,基底层细胞部分缺失,上皮表面细胞损伤脱落,靠近角膜表面逐渐变成鳞状上皮细胞。结膜上皮出现过度增生,角化增加,杯状细胞分布更靠近基底层细胞。HRS滴眼组和HRS腹腔注射组大鼠角结膜上皮均变平整规则,细胞层次减少,边界清晰,水肿减轻,组织增生减少。5.PAS染色:光镜下观察,干眼组大鼠结膜杯状细胞大小不均匀,靠近基底部,个数减少。HRS滴眼组和HRS腹腔注射组均可见大鼠结膜杯状细胞大小趋于正常,个数增多。比较两组之间杯状细胞计数,差异无明显统计学意义(P0.05)。6.TUNEL检测:荧光显微镜下观察,干眼组大鼠角结膜上皮凋亡细胞较正常对照组明显增多。HRS滴眼组和HRS腹腔注射组大鼠角膜上皮凋亡细胞较干眼组和NS干预组明显减少,细胞大小均匀,表层光滑。比较两组之间凋亡细胞计数,差异无明显统计学意义(P0.05)。7.免疫组化染色:正常组大鼠角膜、睑结膜上皮均未见K10表达,穹隆部结膜可见K10弱表达。干眼组大鼠角膜上皮和穹隆部结膜K10表达强阳性,睑结膜未见明显K10表达。HRS滴眼组和HRS腹腔注射组大鼠角膜、穹隆部结膜上皮均K10表达明显减弱。8.免疫印记:干眼组角结膜组织NF-kB较正常组表达明显增多,HRS滴眼组和HRS腹腔注射组大鼠角结膜组织NF-kB表达较干眼组和NS干预组减少,且两组间差异不具统计学意义(P0.05)。结论本实验采用HRS滴眼及腹腔注射两种途径干预东莨菪碱诱导的大鼠干眼模型,通过检测各组大鼠干眼的临床指标泪液分泌试验、泪膜破裂时间、角膜上皮荧光素钠染色评分,角结膜组织学病理改变、细胞凋亡情况、鳞状上皮化生程度以及炎症因子NF-kB的表达,发现HRS滴眼和腹腔注射均能改善干眼体征;使角结膜上皮增生减少,杯状细胞形态趋于正常;角结膜上皮凋亡细胞明显减少;角结膜鳞状上皮化生改善;角结膜组织NF-kB表达减少。综上所述,HRS可能通过抑制炎症反应来缓解东莨菪碱诱导的大鼠干眼眼表损伤,对干眼具有保护作用,有望成为治疗干眼的新型药物。
[Abstract]:Dry eye (DE) is one of the common diseases of the ocular surface. It is mainly manifested in ocular discomfort, decreased visual quality, increased lacrimal osmotic pressure and ocular surface inflammation, inflammation, imbalance of sex hormone secretion, dysfunction of nerve, apoptosis and oxidative stress in the pathogenesis of dry eyes. Effect, but easy to relapse after drug withdrawal. Local application of anti inflammatory drugs can inhibit inflammatory factors and alleviate dry eye symptoms, but long-term use of toxic and side effects. How to effectively control and even reverse the dry eye, looking for a safe and effective drug is imminent. The hydrogen rich saline (hydrogen-rich saline, HRS) was dissolved in the normal saline at 0.4MPa high pressure and reached the saturation concentration of 0.6mmol/L. It showed obvious anti-inflammatory effects in the animal models of various diseases. This experiment used two ways to interfere with the dry eye model of scopolamine induced rats by HRS eye drops and intraperitoneal injection. Type, the clinical indexes of rats, the pathological changes of the conjunctiva, the apoptosis, the degree of squamous metaplasia and the expression of the -kB (nuclear factor-kB, NF-kB) of the inflammatory related factors were detected to investigate the protective effect of HRS on the ocular surface of the dry eye of the rats. Square method 1. used the random digital table method to make 30 healthy Wistar of 6 weeks old. The rats were divided into 6 groups: normal group (A), dry eye group (B), HRS eye drop group (C), physiological saline (normal saline, NS) eye drop group (D), HRS intraperitoneal injection group (E), NS peritoneal injection group (F), 5 in each group (10 eyes). The latter five groups of hind limbs were subcutaneously injected with scopolamine hydrobromide to establish dry eye model, 4 times every day (9 points, 12 points, 15 points, 18 points), each time, The left and right sides were alternately carried out. The C and D groups were treated with HRS, NS, 1 times per hour and 9 times a day (8:30 -16 and a half); E and F groups were intraperitoneally injected with HRS, NS 1 times (18:30) per day, respectively, for 28 days before and after the medication, respectively. The tear film rupture time (break-up time, BUT) and corneal epithelial fluorescein sodium staining score were recorded, and 28 days after.3. were recorded, the rats were killed by the dislocation of the cervical vertebra. The whole eyeball was taken and the paraffin section was stained with HE. The histological changes of the conjunctiva in each group were observed under light microscope and.4. was stained by iodic acid Schiff (periodic acid Schiff stain, PAS) and under light microscope. The morphology of the conjunctival goblet epithelial cells in each group was observed and the number of.5. in situ end transferase labeling (TUNEL) was counted under the light microscope. The apoptosis of the conjunctiva in each group was detected under the fluorescence microscope, and the number of apoptotic cells of each high field of visual field under the fluorescence microscope was counted by immunohistochemical staining and observed under light microscope. The expression of keratin 10 (keratin 10, K10) in the angular conjunctiva of rats and the evaluation of the squamous metaplasia.7. to take the conjunctival tissue of rats in each group. The expression of NF-kB in the conjunctiva of each group was detected by immuno imprint. Results 1.S I t: was statistically significant (F=2.897, P0.05), and dry eyes (F=2.897, P0.05), and dry eyes compared with each group of rats in each group for 7 days (F=2.897, P0.05). Compared with the normal group, the value of SIt was lower than that of the normal group (P0.05). The difference of S i t value in the 14 days of model rats was statistically significant (F=5.194, P0.05), S i t values in HRS eye drop group and HRS intraperitoneal injection group were higher than those in dry eye group (P0.05). There was no significant difference in S i t value between group and HRS intraperitoneal injection group (P0.05). After 28 days, it remained stable.2.BUT:, and the difference of BUT in each group was statistically significant (F=2.910, P0.05), and the BUT values in the dry eye group decreased compared with the normal group (P0.05). The difference between the 14 days BUT of rats in each group was statistically significant (F=3.894, respectively). The BUT values in the HRS eye drop group and the HRS intraperitoneal injection group were all higher than those in the dry eye group (P0.05), and the BUT values in the HRS eye drop group and the HRS intraperitoneal group were higher than those in the NS drops and the NS intraperitoneal injection group (P0.05), and the difference between the HRS eye drop group and the HRS intraperitoneal injection group was not statistically significant. The fluorescein sodium staining in the corneal epithelium remained stable to the 28 day. Color score: the difference of fluorescein sodium staining score of corneal epithelium in each group was statistically significant (F=2.424, P0.05) for 7 days. The staining score of dry eye group was higher than that of the normal group (P0.05). The difference of fluorescein sodium staining score in corneal epithelium of rats in each group was statistically significant (F=16.487, P0.05) and HRS eye drop group. The staining scores of HRS intraperitoneal injection group were lower than those in the dry eye group (P0.05), and the staining scores of HRS eye drop group and HRS intraperitoneal injection group were lower than those of NS eye drop group and NS intraperitoneal injection group (P0.05), and the difference between HRS drop eye group and HRS intraperitoneal injection group was not statistically significant (P0.05). To the 28 day, the stable.4.HE staining was maintained: light microscope view The corneal epithelial cells in the dry eye group were blurred, the cells were not arranged closely, the cell volume was large, the cells in the basal layer were missing, the epithelial cells were damaged and shedding and the epithelial cells were gradually transformed into squamous cells near the corneal surface. The conjunctival epithelium was hyperplastic and the keratinization was increased. The distribution of goblet cells was closer to the.HRS eye drops and the HRS abdominal cavity in the basal layer cells. The cornea conjunctival epithelium of the rats in the injection group became smooth and smooth, the cell level was reduced, the boundary was clear, the edema was reduced, and the tissue proliferation was reduced by.5.PAS staining. The size of the conjunctival goblet cells in the dry eye group was not uniform. The size of the conjunctival goblet cells in the.HRS eye group and the HRS intraperitoneal injection group were close to the base, and the size of the conjunctival goblet cells tended to tend to be more and more obvious. The count of goblet cells between the two groups was compared. There was no significant statistical significance (P0.05).6.TUNEL detection: the apoptotic cells in the cornea conjunctiva epithelium of the dry eye group were significantly higher than those in the normal control group. The apoptotic cells in the.HRS eye group and the HRS intraperitoneal injection group were compared with those of the dry eye group and the NS intervention group. The cell size was uniform and the surface layer was smooth. The number of apoptotic cells between the two groups was compared. The difference was not statistically significant (P0.05).7. immunohistochemical staining: there was no K10 expression in the cornea and the conjunctiva epithelium of the normal group, and the weak expression of K10 in the conjunctiva of the dome. The expression of K10 in the corneal epithelium and the conjunctiva of the dome of the dry eye group was strongly positive, and the palpebral knot was found. There was no obvious K10 expression in the.HRS eye group and HRS intraperitoneal injection group, and the expression of K10 in the conjunctival epithelium of the dome was significantly weakened by.8. immuno imprint. The expression of NF-kB in the conjunctiva tissue in the dry eye group was significantly higher than that in the normal group. The NF-kB expression in the cornea conjunctiva tissue of the HRS eye drop group and HRS intraperitoneal injection group was less than that in the dry eye group and the NS intervention group, and the two groups were reduced. The difference was not statistically significant (P0.05). Conclusion the experiment was carried out by using HRS eye drops and intraperitoneal injection to interfere with the dry eye model induced by scopolamine. The tear secretion test, tear film rupture time, corneal epithelial fluorescein sodium staining score, and histopathological changes of cornea conjunctiva were detected in the dry eyes of rats in each group by two kinds of intraperitoneal injection. Apoptosis, squamous metaplasia and expression of inflammatory factor NF-kB showed that HRS eye drops and intraperitoneal injection can improve dry eye signs, reduce the hyperplasia of the conjunctiva epithelium, the shape of goblet cells tend to be normal, the apoptotic cells in the conjunctival epithelium of the angular conjunctiva obviously decrease; the angular conjunctiva squamous epithelium improves; the expression of NF-kB in the conjunctiva is reduced. As mentioned above, HRS may alleviate the damage of scopolamine induced dry eye ocular surface by inhibiting the inflammatory response, and it has a protective effect on dry eyes and is expected to be a new drug for the treatment of dry eyes.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R777.34;R-332

【参考文献】