發(fā)布時間：2018-04-21 10:02

  本文選題：角膜緣干細胞缺乏 + 堿燒傷�。� 參考：《青島大學(xué)》2013年博士論文

【摘要】：目的 體外培養(yǎng)的異體角膜緣上皮細胞移植術(shù)是治療角膜緣干細胞缺乏的有效方案,在臨床中我們觀察到術(shù)后有些患者會發(fā)生再次角膜新生血管化和角膜上皮反復(fù)缺損,考慮其原因可能為手術(shù)后供體細胞不能很好的存活并發(fā)揮功能。因此,本研究的目的是通過動物模型模擬該手術(shù)及術(shù)后的臨床過程,探討手術(shù)后供體細胞的存活狀態(tài)和其可能的影響因素,并進一步探討手術(shù)后可維持眼表穩(wěn)定的可行措施。 方法 1、動物模型的建立和評價 采用4周齡雌性新西蘭大白兔45只,建立堿燒傷致兔眼角膜緣干細胞缺乏的動物模型。將實驗兔隨機分為2組：中度組和重度組,每組22只,每只兔均選右眼建模、手術(shù)。用內(nèi)徑12mm,外徑18mm的環(huán)形濾紙片浸透1mol/L NaOH,覆蓋于實驗兔角膜緣處,重度組接觸1min,中度組接觸30s。然后用0.9%氯化鈉注射液沖洗角膜表面和結(jié)膜囊5分鐘。燒傷后每天觀察兔角膜變化,并于堿燒傷后穩(wěn)定期對角膜新生血管程度和炎癥程度進行評分。用HE染色方法觀察比較正常兔角膜和堿燒傷后穩(wěn)定期兔角膜的組織病理學(xué)差異；用免疫組織化學(xué)方法檢測正常組和堿燒傷后穩(wěn)定期時兔角膜中p63、CD68的表達情況；用Real time-PCR方法檢測正常組和堿燒傷后穩(wěn)定期時兔角膜中干細胞相關(guān)因子ANp63α、ABCG2、CK3,及炎癥因子IL-1β、IL-6、IL-4、IL-10、IL-13、TNF-α、MCP-1、iNOS、TGF-β、 VEGF的表達量。 2、以羊膜為載體人角膜緣上皮細胞體外培養(yǎng) 角膜來自山東省眼科研究所眼庫,取用角膜移植后剩余的角鞏膜環(huán),將角膜上皮細胞消化成單細胞后接種于去上皮羊膜上,每天觀察細胞生長情況,待細胞鋪滿羊膜形成復(fù)層時進行移植手術(shù) 3、以羊膜為載體培養(yǎng)的人角膜緣上皮細胞移植術(shù) 取堿燒傷后處于穩(wěn)定期的兔角膜進行手術(shù)。術(shù)中剪開全周球結(jié)膜,剝除角膜表而覆蓋的血管膜,將培養(yǎng)有人角膜緣上皮細胞的羊膜覆蓋于角膜表面,將羊膜于角膜緣處縫合5～8針固定于球結(jié)膜和鞏膜之間。手術(shù)中注意保護細胞避免受到損傷,術(shù)畢行瞼裂縫合避免角膜過度暴露。 4、手術(shù)后的臨床療效評價和供體細胞的存活狀態(tài)檢測 分別于術(shù)后即刻,術(shù)后1天、3天、7天、14天、21天和28天時用裂隙燈顯微鏡觀察術(shù)后兔角膜變化,對角膜新生血管化程度和炎癥程度進行評分,并利用裂隙燈照相系統(tǒng)進行圖像記錄。在上述各時間點取兔角膜進行HE染色,并用免疫組化方法檢測供體和受體中p63、Ki-67的表達情況。用RT-PCR方法檢測術(shù)前及術(shù)后各時間點供體和受體細胞中干細胞相關(guān)因子ANp63α、Ki-67、ABCG2、C/EBPA、CK3的表達量。 5、手術(shù)后眼表微環(huán)境中炎癥因子表達情況檢測 分別于手術(shù)后即刻、術(shù)后1天、3天、7天、14天、28天時取兔角膜,用免疫組化方法檢測CD68的表達情況,用Real time-PCR方法檢測炎癥因子IL-1β、IL-6、IL-4、IL-10、IL-13、TNF-α、MCP-1、iNOS、TGF-β、VEGF、CD4、CD8的表達量。 結(jié)果 1、堿燒傷后兔角膜的病變特征 堿燒傷后燒傷部位的角膜即呈瓷白色混濁,堿燒傷后1周內(nèi)為急性期,球結(jié)膜缺血,角膜水腫,角膜上皮持續(xù)缺損,堿燒傷后第2-3周進入損傷和修復(fù)共存期。中度堿燒傷組在燒傷后4-8周時角膜新生血管化,炎癥反應(yīng)趨于穩(wěn)定；重度堿燒傷組在傷后8-12周時才能進入穩(wěn)定期。中度堿燒傷組有3眼評分未達標(biāo)而排除研究,重度堿燒傷組3眼角膜穿孔排除研究。根據(jù)評分標(biāo)準(zhǔn),中度組和重度組的總分平均值分別為5.7±1.2分和8.8±1.1分,兩組差別有統(tǒng)計學(xué)意義(P=0.000)。HE染色可見堿燒傷后穩(wěn)定期時角膜表面覆蓋扁平的鱗狀上皮細胞,基質(zhì)內(nèi)見大量管徑粗大的新生血管,可達深基質(zhì)層。免疫組織化學(xué)檢測可見大量CD68陽性的炎癥細胞浸潤,角膜中p63表達陰性。Real time-PCR結(jié)果顯示堿燒傷組ANp63α、ABCG2、CK3的表達顯著低于正常對照組,其中重度堿燒傷組降低較中度堿燒傷組降低更為顯著(P0.05)。堿燒傷后穩(wěn)定期角膜中炎癥因子IL-1β、 IL-6、IL-4、IL-10、IL-13、TNF-α、 iNOS、MCP-1、TGF-β、VEGF的表達顯著高于正常對照組。 2、以羊膜為載體培養(yǎng)的兔角膜緣上皮細胞移植術(shù)后的臨床療效 中度堿燒傷組在術(shù)后1-28天時均可見到角膜上皮完整,未見新生血管再次生長。重度堿燒傷組在術(shù)后1天時可見到角膜上皮片狀缺損,術(shù)后3天時角膜上皮修復(fù)完整,術(shù)后14天和21天時再次出現(xiàn)角膜上皮片狀缺損,自術(shù)后14天時可見新生學(xué)管再次長入角膜,甚至比術(shù)前更為嚴(yán)重。HE染色可見術(shù)后7天內(nèi)羊膜與角膜貼附不緊密,術(shù)后14天時羊膜吸收,但此時角膜上皮細胞與角膜基質(zhì)仍有分離現(xiàn)象。中度堿燒傷組術(shù)后21天和28天時角膜上皮完整,角膜基質(zhì)內(nèi)未見新生血管管腔；重度堿燒傷組可見角膜上皮缺損,或角膜上皮不規(guī)則增生,呈鱗狀上皮化,角膜基質(zhì)內(nèi)大量炎癥細胞浸澗,并可見到新生血管管腔。 3、手術(shù)后供體細胞的存活狀態(tài) 免疫組織化學(xué)檢測在中度堿燒傷組術(shù)后28天內(nèi)均可見供體p63和Ki-67表達。重度堿燒傷組術(shù)后7天內(nèi)可見p63表達,偶見Ki-67陽性細胞,RT-PCR結(jié)果顯示兩組供體細胞中ANp63α的農(nóng)達在手術(shù)后即刻降低約40%,術(shù)后3天時顯著降低,而ABCG2在術(shù)后28天內(nèi)均有表達但呈逐漸下降趨勢。中度堿燒傷組Ki-67的表達量在術(shù)后7天內(nèi)較術(shù)前升高,但術(shù)后14天時即顯著降低至消失；C/EBPA和CK3的表達量在術(shù)后即刻較術(shù)前降低,但術(shù)后1天時有所上調(diào),術(shù)后3天、7天時呈顯著下降,至術(shù)后14天后幾乎檢測不到其表達。重度堿燒傷組Ki-67、C/EBP△、CK3的表達在術(shù)后3天時即顯著降低,術(shù)后7天時表達量極低甚至消失。 4、手術(shù)后眼表炎癥微環(huán)境中炎癥因子的表達 HE染色和免疫組織化學(xué)結(jié)果：術(shù)后1天和3天時在羊膜下和角膜基質(zhì)中可見分葉清晰的中性粒細胞浸潤和CD68陽性的巨噬細胞浸潤,術(shù)后7天時較少見到分葉清晰的中性粒細胞,但CD68陽性細胞仍較多。中度堿燒傷組術(shù)后14天后CD68陽性細胞逐漸減少,而重度堿燒傷組CD68陽性細胞浸潤可持續(xù)至術(shù)后28天。 Real-time PCR結(jié)果：炎癥因子IL-1β、IL-6、MCP-1、IL-13、IL-10的表達在術(shù)后即刻即顯著上調(diào),術(shù)后3天時顯著下調(diào),術(shù)后7天和14天再次上調(diào),隨后逐漸下降。大部分炎癥因子在術(shù)后出現(xiàn)兩個高峰,即術(shù)后即刻和術(shù)后7天時。CD4和CD8的表達在術(shù)后1天、3天均處在極低水平,術(shù)后7天時驟然上調(diào),此后下降。 5、手術(shù)后受體中干細胞相關(guān)因子的表達 中度堿燒傷組ANp63α、ABCG2、CK3、Ki-67的表達在術(shù)后較術(shù)前呈上調(diào)趨勢,在術(shù)后21天時較為顯著。重度堿燒傷組ANp63α、ABCG2、CK3、Ki-67在術(shù)后的表達量沒有超過術(shù)前,在術(shù)后早期如1天、3天、7天時甚至較術(shù)前降低。 結(jié)論 1、角膜緣環(huán)形堿燒傷是建立角膜緣干細胞缺乏模型的有效方法,在堿燒傷后穩(wěn)定期角膜內(nèi)仍有大量巨噬細胞浸潤和多種炎癥因子高表達,中度堿燒傷角膜緣內(nèi)可能殘存一些可恢復(fù)功能的干細胞。 2、移植的供體細胞在術(shù)后7天內(nèi)可維持其增殖、分化和自我更新能力,隨后細胞的各項功能均下降至喪失。 3、術(shù)后早期的炎癥反應(yīng)和免疫排斥反應(yīng)是影響移植的供體細胞功能的關(guān)鍵因素,巨噬細胞可能在其中發(fā)揮了主要作用。 4、有足夠的供體細胞長期存活并發(fā)揮正常功能是術(shù)后維持穩(wěn)定眼表的一個必要條件,而受體自身殘存足夠的可恢復(fù)功能的角膜緣干細胞是術(shù)后維持穩(wěn)定眼表的另一個重要條件。
[Abstract]:Purpose

The purpose of this study was to investigate the survival status of donor cells and the possible influencing factors after operation , and to further explore the feasible measures to maintain ocular surface stability after operation .

method

1 . Establishment and evaluation of animal model

The experimental rabbits were randomly divided into 2 groups : moderate group and severe group .
The expression of p63 and CD68 in rabbit cornea was detected by immunohistochemistry .
The expression of ANp63 偽 , ABCG2 , CK3 , and inflammatory cytokines IL - 1尾 , IL - 6 , IL - 4 , IL - 10 , IL - 13 , TNF - 偽 , MCP - 1 , iNOS , TGF - 尾 and VEGF were detected by Real time - PCR .

2 . In vitro culture of limbal epithelial cells with amniotic membrane as carrier

The corneal epithelial cells were digested into single cells and seeded on the deepithelialized amniotic membrane after corneal transplantation , and the cell growth was observed every day , and the transplantation was carried out when the cells were covered with the amniotic membrane to form a complex layer .

3 . Human corneal epithelial cell transplantation with amniotic membrane as carrier

After taking alkali burn , the rabbits were operated in a stable rabbit cornea . The whole circumference bulbar conjunctiva was cut off and the corneal surface was peeled off . The amniotic membrane cultured with corneal limbal epithelial cells was covered on the surface of the cornea , and the amniotic membrane was sutured between the conjunctiva and sclera at the corneal margin .

4 . Evaluation of clinical efficacy after surgery and test for survival status of donor cells

The expressions of p63 , Ki - 67 , ABCG2 , C / EBPA , CK3 in donor and recipient cells were detected by RT - PCR . The expression of p63 , Ki - 67 , ABCG2 , C / EBPA and CK3 in donor and recipient cells were detected by RT - PCR .

5 . Detection of inflammatory factor expression in ocular surface microenvironment after operation

The expression of IL - 1尾 , IL - 6 , IL - 4 , IL - 10 , IL - 13 , TNF - 偽 , MCP - 1 , iNOS , TGF - 尾 , VEGF , CD4 and CD8 were measured by Real time - PCR . The expression of IL - 1尾 , IL - 6 , IL - 4 , IL - 10 , IL - 13 , TNF - 偽 , MCP - 1 , iNOS , TGF - 尾 , VEGF , CD4 , CD8 were measured by Real time - PCR .

Results

The pathological characteristics of rabbit cornea after alkali burn

The corneal neovascularization and inflammatory response were stable at 4 - 8 weeks after burn .
The expression of IL - 1尾 , IL - 6 , IL - 4 , IL - 10 , IL - 13 , TNF - 偽 , iNOS , MCP - 1 , TGF - 尾 and VEGF in patients with severe alkali burn were significantly higher than those in control group .

2 . Clinical efficacy of amniotic membrane as carrier in rabbit corneal epithelial cell transplantation

The corneal epithelium integrity was observed at 1 - 28 days after operation . Corneal epithelium flap defects were seen at 1 day after operation . The corneal epithelium was completely repaired at 1 day after operation . The corneal epithelium was re - inserted into the cornea at 14 days after operation . The corneal epithelium was absorbed at 14 days after operation .
In severe alkali burn group , corneal epithelial defect , corneal epithelium irregular hyperplasia , squamous epithelization , large amount of inflammatory cells in corneal stroma are immersed in the stream , and the neovascular lumen can be seen .

3 . Survival status of donor cells after surgery

The expression of p63 and Ki - 67 were observed within 28 days after operation of moderate alkaline burn group . The expression of Ki - 67 was significantly decreased in the medium - base burn group after operation , but the expression of Ki - 67 in the medium - base burn group increased significantly within 7 days after operation , but decreased to disappear at 14 days after operation .
The expression of C / EBPA and CK3 decreased immediately before operation , but was up - regulated at 1 day after operation . The expression of C / EBPA and CK3 was significantly decreased at 7 days after operation . The expression of Ki - 67 , C / EBP 鈻，

本文編號：1781955