本文選題：角膜緣干細胞缺乏 + 堿燒傷��； 參考：《青島大學》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缺損,考慮其原因可能為手術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及術后的臨床過程,探討手術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。用內徑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差異；用免疫組織化學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層時進行移植手術 3、以羊膜為載體培養(yǎng)的人角膜緣上皮細胞移植術 取堿燒傷后處于穩(wěn)定期的兔角膜進行手術。術中剪開全周球結膜,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針固定于球結膜和鞏膜之間。手術中注意保護細胞避免受到損傷,術畢行瞼裂縫合避免角膜過度暴露。 4、手術后的臨床療效評價和供體細胞的存活狀態(tài)檢測 分別于術后即刻,術后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方法檢測術前及術后各時間點供體和受體細胞中干細胞相關因子ANp63α、Ki-67、ABCG2、C/EBPA、CK3的表達量。 5、手術后眼表微環(huán)境中炎癥因子表達情況檢測 分別于手術后即刻、術后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周內為急性期,球結膜缺血,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)評分標準,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,基質內見大量管徑粗大的新生血管,可達深基質層。免疫組織化學檢測可見大量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后的臨床療效 中度堿燒傷組在術后1-28天時均可見到角膜上皮完整,未見新生血管再次生長。重度堿燒傷組在術后1天時可見到角膜上皮片狀缺損,術后3天時角膜上皮修復完整,術后14天和21天時再次出現(xiàn)角膜上皮片狀缺損,自術后14天時可見新生學管再次長入角膜,甚至比術前更為嚴重。HE染色可見術后7天內羊膜與角膜貼附不緊密,術后14天時羊膜吸收,但此時角膜上皮細胞與角膜基質仍有分離現(xiàn)象。中度堿燒傷組術后21天和28天時角膜上皮完整,角膜基質內未見新生血管管腔；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,角膜基質內大量炎癥細胞浸澗,并可見到新生血管管腔。 3、手術后供體細胞的存活狀態(tài) 免疫組織化學檢測在中度堿燒傷組術后28天內均可見供體p63和Ki-67表達。重度堿燒傷組術后7天內可見p63表達,偶見Ki-67陽性細胞,RT-PCR結果顯示兩組供體細胞中ANp63α的農(nóng)達在手術后即刻降低約40%,術后3天時顯著降低,而ABCG2在術后28天內均有表達但呈逐漸下降趨勢。中度堿燒傷組Ki-67的表達量在術后7天內較術前升高,但術后14天時即顯著降低至消失；C/EBPA和CK3的表達量在術后即刻較術前降低,但術后1天時有所上調,術后3天、7天時呈顯著下降,至術后14天后幾乎檢測不到其表達。重度堿燒傷組Ki-67、C/EBP△、CK3的表達在術后3天時即顯著降低,術后7天時表達量極低甚至消失。 4、手術后眼表炎癥微環(huán)境中炎癥因子的表達 HE染色和免疫組織化學結果：術后1天和3天時在羊膜下和角膜基質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)至術后28天。 Real-time PCR結果：炎癥因子IL-1β、IL-6、MCP-1、IL-13、IL-10的表達在術后即刻即顯著上調,術后3天時顯著下調,術后7天和14天再次上調,隨后逐漸下降。大部分炎癥因子在術后出現(xiàn)兩個高峰,即術后即刻和術后7天時。CD4和CD8的表達在術后1天、3天均處在極低水平,術后7天時驟然上調,此后下降。 5、手術后受體中干細胞相關因子的表達 中度堿燒傷組ANp63α、ABCG2、CK3、Ki-67的表達在術后較術前呈上調趨勢,在術后21天時較為顯著。重度堿燒傷組ANp63α、ABCG2、CK3、Ki-67在術后的表達量沒有超過術前,在術后早期如1天、3天、7天時甚至較術前降低。 結論 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可能殘存一些可恢復功能的干細胞。 2、移植的供體細胞在術后7天內可維持其增殖、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、術后早期的炎癥反應和免疫排斥反應是影響移植的供體細胞功能的關鍵因素,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功能的角膜緣干細胞是術后維持穩(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