本文選題：大鼠干眼 + 富氫鹽水��； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的干眼(dry eye,DE)是眼表常見疾病之一,主要表現(xiàn)為眼部不適,視覺質(zhì)量下降,伴有淚液滲透壓升高和眼表炎癥。炎癥、性激素分泌失衡、神經(jīng)機(jī)能障礙、細(xì)胞凋亡及氧化應(yīng)激共同參與干眼的發(fā)病機(jī)制。目前,人工淚液治療干眼雖有一定療效,但停藥后易復(fù)發(fā)。局部應(yīng)用抗炎藥物可抑制炎癥因子,減輕干眼癥狀,但長期使用存在毒副作用。如何有效的控制甚至逆轉(zhuǎn)干眼,尋找一種安全有效的藥物迫在眉睫。研究發(fā)現(xiàn),氫氣具選擇性抗氧化作用,同時(shí)它可抑制炎性因子的釋放,具有抗炎作用。富氫鹽水(hydrogen-rich saline,HRS)是將氫氣在0.4MPa高壓下溶于生理鹽水,達(dá)到飽和濃度為0.6mmol/L,它在多種疾病的動(dòng)物模型中均表現(xiàn)出明顯抗炎作用。本實(shí)驗(yàn)使用HRS滴眼及腹腔注射兩種途徑干預(yù)東莨菪堿誘導(dǎo)的大鼠干眼模型,檢測(cè)各組大鼠的臨床指標(biāo)、角結(jié)膜組織病理變化、細(xì)胞凋亡情況、鱗狀上皮化生程度以及炎癥相關(guān)因子核因子-kB(nuclear factor-kB,NF-kB)的表達(dá),探討HRS對(duì)大鼠干眼眼表是否具有保護(hù)作用。方法1.采用隨機(jī)數(shù)字表法將30只6周齡健康Wistar大鼠分為6組,分別為正常組(A)、干眼組(B)、HRS滴眼組(C)、生理鹽水(normal saline,NS)滴眼組(D)、HRS腹腔注射組(E)、NS腹腔注射組(F),每組5只(10眼)。后五組后肢皮下注射3mg·mL-1氫溴酸東莨菪堿建立干眼模型,每天注射4次(9點(diǎn)、12點(diǎn)、15點(diǎn)、18點(diǎn)),每次0.5ml,左右兩側(cè)交替進(jìn)行。對(duì)C組和D組分別用HRS、NS點(diǎn)眼,每小時(shí)點(diǎn)眼1次,每天點(diǎn)眼9次(8點(diǎn)半-16點(diǎn)半);E組和F組按5ml·kg-1每天分別腹腔注射HRS、NS 1次(18點(diǎn)半),連續(xù)28天。2.用藥前及用藥后7、14、21、28天分別對(duì)各組大鼠行淚液分泌試驗(yàn)(SchirmerⅠtest,SⅠt)、淚膜破裂時(shí)間(break-up time,BUT)和角膜上皮熒光素鈉染色評(píng)分,記錄數(shù)據(jù)。3.用藥后28天,頸椎脫臼法處死大鼠,取整個(gè)眼球,石蠟切片后行HE染色,光鏡下觀察各組大鼠角結(jié)膜組織學(xué)變化。4.行過碘酸-雪夫(periodic acid schiff stain,PAS)染色,光鏡下觀察各組大鼠結(jié)膜杯狀上皮細(xì)胞形態(tài),并計(jì)數(shù)光鏡下每高倍視野杯狀細(xì)胞數(shù)目。5.原位末端轉(zhuǎn)移酶標(biāo)記(TUNEL)法,熒光顯微鏡下檢測(cè)各組大鼠角結(jié)膜凋亡情況,并計(jì)數(shù)熒光顯微鏡下每高倍視野細(xì)胞凋亡個(gè)數(shù)。6.行免疫組化染色,光鏡下觀察各組大鼠角結(jié)膜組織中角蛋白10(keratin 10,K10)的表達(dá),評(píng)估鱗狀上皮化生程度。7.取各組大鼠角結(jié)膜組織,采用免疫印記法檢測(cè)各組大鼠角結(jié)膜組織NF-kB的表達(dá)。結(jié)果1.SⅠt:比較各組大鼠造模7天的SⅠt差異具有統(tǒng)計(jì)學(xué)意義(F=2.897,P0.05),干眼組SIt值與正常組相比下降(P0.05)。比較各組大鼠造模14天SⅠt值差異具有統(tǒng)計(jì)學(xué)意義(F=5.194,P0.05),HRS滴眼組和HRS腹腔注射組SⅠt值均比干眼組升高(P0.05);HRS滴眼組和HRS腹腔注射組SⅠt值分別比NS滴眼組和NS腹腔注射組升高(P0.05);且HRS滴眼組和HRS腹腔注射組SⅠt值差異始終無統(tǒng)計(jì)學(xué)意義(P0.05)。至28天始終保持穩(wěn)定。2.BUT:比較各組大鼠造模7天BUT差異具有統(tǒng)計(jì)學(xué)意義(F=2.910,P0.05),干眼組BUT值與正常組相比下降(P0.05)。比較各組大鼠造模14天BUT差異具有統(tǒng)計(jì)學(xué)意義(F=3.894,P0.05),HRS滴眼組和HRS腹腔注射組BUT值均比干眼組升高(P0.05);HRS滴眼組和HRS腹腔注射組BUT值分別比NS滴眼組和NS腹腔注射組升高(P0.05);且HRS滴眼組和HRS腹腔注射組BUT值差異始終無統(tǒng)計(jì)學(xué)意義(P0.05)。至28天始終保持穩(wěn)定。3.角膜上皮熒光素鈉染色評(píng)分:比較各組大鼠造模7天的角膜上皮熒光素鈉染色評(píng)分差異具有統(tǒng)計(jì)學(xué)意義(F=2.424,P0.05),干眼組染色評(píng)分與正常組相比升高(P0.05)。比較各組大鼠造模14天的角膜上皮熒光素鈉染色評(píng)分差異具有統(tǒng)計(jì)學(xué)意義(F=16.487,P0.05),HRS滴眼組和HRS腹腔注射組染色評(píng)分均比干眼組下降(P0.05);HRS滴眼組和HRS腹腔注射組染色評(píng)分分別比NS滴眼組和NS腹腔注射組下降(P0.05);且HRS滴眼組和HRS腹腔注射組染色評(píng)分差異始終無統(tǒng)計(jì)學(xué)意義(P0.05)。至28天始終保持穩(wěn)定。4.HE染色:光鏡下觀察,干眼組角膜上皮細(xì)胞分層模糊,排列不緊密,細(xì)胞體積較大,基底層細(xì)胞部分缺失,上皮表面細(xì)胞損傷脫落,靠近角膜表面逐漸變成鱗狀上皮細(xì)胞。結(jié)膜上皮出現(xiàn)過度增生,角化增加,杯狀細(xì)胞分布更靠近基底層細(xì)胞。HRS滴眼組和HRS腹腔注射組大鼠角結(jié)膜上皮均變平整規(guī)則,細(xì)胞層次減少,邊界清晰,水腫減輕,組織增生減少。5.PAS染色:光鏡下觀察,干眼組大鼠結(jié)膜杯狀細(xì)胞大小不均勻,靠近基底部,個(gè)數(shù)減少。HRS滴眼組和HRS腹腔注射組均可見大鼠結(jié)膜杯狀細(xì)胞大小趨于正常,個(gè)數(shù)增多。比較兩組之間杯狀細(xì)胞計(jì)數(shù),差異無明顯統(tǒng)計(jì)學(xué)意義(P0.05)。6.TUNEL檢測(cè):熒光顯微鏡下觀察,干眼組大鼠角結(jié)膜上皮凋亡細(xì)胞較正常對(duì)照組明顯增多。HRS滴眼組和HRS腹腔注射組大鼠角膜上皮凋亡細(xì)胞較干眼組和NS干預(yù)組明顯減少,細(xì)胞大小均勻,表層光滑。比較兩組之間凋亡細(xì)胞計(jì)數(shù),差異無明顯統(tǒng)計(jì)學(xué)意義(P0.05)。7.免疫組化染色:正常組大鼠角膜、瞼結(jié)膜上皮均未見K10表達(dá),穹隆部結(jié)膜可見K10弱表達(dá)。干眼組大鼠角膜上皮和穹隆部結(jié)膜K10表達(dá)強(qiáng)陽性,瞼結(jié)膜未見明顯K10表達(dá)。HRS滴眼組和HRS腹腔注射組大鼠角膜、穹隆部結(jié)膜上皮均K10表達(dá)明顯減弱。8.免疫印記:干眼組角結(jié)膜組織NF-kB較正常組表達(dá)明顯增多,HRS滴眼組和HRS腹腔注射組大鼠角結(jié)膜組織NF-kB表達(dá)較干眼組和NS干預(yù)組減少,且兩組間差異不具統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論本實(shí)驗(yàn)采用HRS滴眼及腹腔注射兩種途徑干預(yù)東莨菪堿誘導(dǎo)的大鼠干眼模型,通過檢測(cè)各組大鼠干眼的臨床指標(biāo)淚液分泌試驗(yàn)、淚膜破裂時(shí)間、角膜上皮熒光素鈉染色評(píng)分,角結(jié)膜組織學(xué)病理改變、細(xì)胞凋亡情況、鱗狀上皮化生程度以及炎癥因子NF-kB的表達(dá),發(fā)現(xiàn)HRS滴眼和腹腔注射均能改善干眼體征;使角結(jié)膜上皮增生減少,杯狀細(xì)胞形態(tài)趨于正常;角結(jié)膜上皮凋亡細(xì)胞明顯減少;角結(jié)膜鱗狀上皮化生改善;角結(jié)膜組織NF-kB表達(dá)減少。綜上所述,HRS可能通過抑制炎癥反應(yīng)來緩解東莨菪堿誘導(dǎo)的大鼠干眼眼表損傷,對(duì)干眼具有保護(hù)作用,有望成為治療干眼的新型藥物。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R777.34;R-332

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