CAJ | 학술논문

背景角膜组织工程生物材料在体内应当具备透明性、一定的机械强度、生物相容性和缓慢降解的特点，丝素膜生物材料具备这些特征。目的研究采用物理交联再生丝素膜构建组织工程角膜的可行性。方法用常规培养法培养人角膜上皮细胞( CECs)，将对数生长期的CECs在用家蚕丝制备的物理交联再生丝素膜上进行培养，分别于培养后24、48、72 h在倒置显微镜、扫描电子显微镜下观察CECs的生长状态和形态学，并与细胞培养板培养的人CECs形态进行对照。MTT法每日检测和记录再生丝素膜培养后人CECs的增生情况，流式细胞仪检测再生丝素膜培养人CECs的细胞周期和凋亡率。将再生丝素膜4 mm×3 mm植入新西兰白兔右眼角膜基质层间，于术后1个月、2个月时裂隙灯下检查眼表，术后2个月摘除兔眼并制备移植眼角膜组织切片行组织病理学检查，观察再生丝素膜材料的降解情况及角膜新生血管(CNV)的生长情况。采用免疫组织化学法检测术后1个月、2个月CD34在移植眼角膜组织中的表达，并与正常眼进行对照。结果在24、48、72h3个时间点，倒置显微镜、扫描电子显微镜下见再生丝素膜培养的人CECs与细胞培养板培养法培养的人CECs形态结构无明显差别。再生丝素膜或细胞培养板培养CECs在1、2、3、4、5、6、7d各个时间点A490值比较，差异无统计学意义(F=0.641，P＞0.05)。再生丝素膜和细胞培养板培养的CECs凋亡率分别为1.8％、2.0％，细胞G2/G1期分别为1.956、1.945。再生丝素膜角膜植入后2个月时，再生丝素膜为排列整齐的胶原组织替代，角膜内新生血管和炎性细胞均少于移植后1个月。角膜免疫组织化学染色检测显示再生丝素膜植入角膜后1个月、2个月CD34表达量均明显低于Ad-VEGF165诱导的阳性对照，而与正常的角膜相比差异无统计学意义(P＞0.05)。术后1个月与术后2个月比较，角膜中CD34阳性率的差异无统计学意义(P＞0.05)。结论物理交联再生丝素膜构建组织工程角膜可行，再生丝素膜与角膜组织生物相容性良好，再生丝素膜植入兔角膜基质后无明显的炎症反应和新生血管。揹景角膜組織工程生物材料在體內應噹具備透明性、一定的機械彊度、生物相容性和緩慢降解的特點，絲素膜生物材料具備這些特徵。目的研究採用物理交聯再生絲素膜構建組織工程角膜的可行性。方法用常規培養法培養人角膜上皮細胞( CECs)，將對數生長期的CECs在用傢蠶絲製備的物理交聯再生絲素膜上進行培養，分彆于培養後24、48、72 h在倒置顯微鏡、掃描電子顯微鏡下觀察CECs的生長狀態和形態學，併與細胞培養闆培養的人CECs形態進行對照。MTT法每日檢測和記錄再生絲素膜培養後人CECs的增生情況，流式細胞儀檢測再生絲素膜培養人CECs的細胞週期和凋亡率。將再生絲素膜4 mm×3 mm植入新西蘭白兔右眼角膜基質層間，于術後1箇月、2箇月時裂隙燈下檢查眼錶，術後2箇月摘除兔眼併製備移植眼角膜組織切片行組織病理學檢查，觀察再生絲素膜材料的降解情況及角膜新生血管(CNV)的生長情況。採用免疫組織化學法檢測術後1箇月、2箇月CD34在移植眼角膜組織中的錶達，併與正常眼進行對照。結果在24、48、72h3箇時間點，倒置顯微鏡、掃描電子顯微鏡下見再生絲素膜培養的人CECs與細胞培養闆培養法培養的人CECs形態結構無明顯差彆。再生絲素膜或細胞培養闆培養CECs在1、2、3、4、5、6、7d各箇時間點A490值比較，差異無統計學意義(F=0.641，P＞0.05)。再生絲素膜和細胞培養闆培養的CECs凋亡率分彆為1.8％、2.0％，細胞G2/G1期分彆為1.956、1.945。再生絲素膜角膜植入後2箇月時，再生絲素膜為排列整齊的膠原組織替代，角膜內新生血管和炎性細胞均少于移植後1箇月。角膜免疫組織化學染色檢測顯示再生絲素膜植入角膜後1箇月、2箇月CD34錶達量均明顯低于Ad-VEGF165誘導的暘性對照，而與正常的角膜相比差異無統計學意義(P＞0.05)。術後1箇月與術後2箇月比較，角膜中CD34暘性率的差異無統計學意義(P＞0.05)。結論物理交聯再生絲素膜構建組織工程角膜可行，再生絲素膜與角膜組織生物相容性良好，再生絲素膜植入兔角膜基質後無明顯的炎癥反應和新生血管。
배경 각막조직공정생물재료재체내응당구비투명성、일정적궤계강도、생물상용성화완만강해적특점，사소막생물재료구비저사특정。 목적 연구채용물리교련재생사소막구건조직공정각막적가행성。방법 용상규배양법배양인각막상피세포( CECs)，장대수생장기적CECs재용가잠사제비적물리교련재생사소막상진행배양，분별우배양후24、48、72 h재도치현미경、소묘전자현미경하관찰CECs적생장상태화형태학，병여세포배양판배양적인CECs형태진행대조。MTT법매일검측화기록재생사소막배양후인CECs적증생정황，류식세포의검측재생사소막배양인CECs적세포주기화조망솔。장재생사소막4 mm×3 mm식입신서란백토우안각막기질층간，우술후1개월、2개월시렬극등하검사안표，술후2개월적제토안병제비이식안각막조직절편행조직병이학검사，관찰재생사소막재료적강해정황급각막신생혈관(CNV)적생장정황。채용면역조직화학법검측술후1개월、2개월CD34재이식안각막조직중적표체，병여정상안진행대조。결과재24、48、72h3개시간점，도치현미경、소묘전자현미경하견재생사소막배양적인CECs여세포배양판배양법배양적인CECs형태결구무명현차별。재생사소막혹세포배양판배양CECs재1、2、3、4、5、6、7d각개시간점A490치비교，차이무통계학의의(F=0.641，P＞0.05)。재생사소막화세포배양판배양적CECs조망솔분별위1.8％、2.0％，세포G2/G1기분별위1.956、1.945。재생사소막각막식입후2개월시，재생사소막위배렬정제적효원조직체대，각막내신생혈관화염성세포균소우이식후1개월。각막면역조직화학염색검측현시재생사소막식입각막후1개월、2개월CD34표체량균명현저우Ad-VEGF165유도적양성대조，이여정상적각막상비차이무통계학의의(P＞0.05)。술후1개월여술후2개월비교，각막중CD34양성솔적차이무통계학의의(P＞0.05)。 결론물리교련재생사소막구건조직공정각막가행，재생사소막여각막조직생물상용성량호，재생사소막식입토각막기질후무명현적염증반응화신생혈관。
Background Biomaterials for corneal tissue engineering must demonstrate several critical features for potential utility in vivo, including transparency, mechanical integrity, biocompatibility and slow biodegradation. Silk film biomaterial had been characterized to meet these functional requirements. Objective This study was to investigate the feasibility of physico-crosslink regenerated silk fibroin film as tissue engineered corneal scaffold. Methods Human corneal epithelial cells(CECs) links were cultured by regular method and CECs in logarithmic phase were than incubated on physico-crosslink regenerated silk fibroin film membrane. The shape of cultured human CECs was observed after 24,48 and 72 hours under the inverted microscope and scanning electron microscope( SEM ) ,and the CECs were cultured on culture plates as controls. The growth state of CECs on regenerated silk fibroin film was observed daily for 7 days by MTT, and cell cycle analysis and the presence of apoptosis of human CECs were examined by flow cytometry after incubation on regenerated silk fibroin film. Regenerated silk fibroin filmCECs (4 mm×3 mm) were implanted into the corneal stroma of the right eyes of New Zealand white rabbits. At the end of 4 and 8 weeks after implantation, the appearance of the ocular surface was examined using slit lamp and corneal neovascular area was measured. Corneal histopathological examination was carried out to assess the degradation of graft materials and immunohistochemistry was performed to detect the expression of CD34 in the corneal tissue after operation. Results The morphology and structure of CECs were identical using the two cultured Methods when observed under the inverted microscope and SEM after 24,48 and 72 hours. No significant difference was found in the A490 value 1,2,3,4,5,6 or 7 days after incubation on regenerated silk membrane and in culture plates ( Fmethod =0. 641 ,P＞0.05 ). The apoptosis rates of CECs on regenerated silk membrane or culture plates were 1.8％ and 2.0％ and the amount of cells in G2/G1 phase was 1. 956 and 1. 945, respectively. Histopathological examination showed that the regenerated silk membrane material degraded and was replaced by regular collagen tissue 2 months after implantation,and the presence of neovascular area and inflammatory cells were less prominent in 2 months than 1 month post-implantation. The expression level of CD34 in corneal tissue was evidently lower 1 and 2 months after operation than the Ad-VEGF165-induced positive control group (P＜0. 05), and no significant differences were seen when compared with normal CECs(P＞0.05). Conclusions Physico- crosslink regenerated silk fibroin film is an excellent biomaterial for tissue engineered corneal scaffold with good biocompatibility.