眼表重建手术后早期供体干细胞的存活状态及其影响因素研究
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摘要
目的
体外培养的异体角膜缘上皮细胞移植术是治疗角膜缘干细胞缺乏的有效方案,在临床中我们观察到术后有些患者会发生再次角膜新生血管化和角膜上皮反复缺损,考虑其原因可能为手术后供体细胞不能很好的存活并发挥功能。因此,本研究的目的是通过动物模型模拟该手术及术后的临床过程,探讨手术后供体细胞的存活状态和其可能的影响因素,并进一步探讨手术后可维持眼表稳定的可行措施。
方法
1、动物模型的建立和评价
采用4周龄雌性新西兰大白兔45只,建立碱烧伤致兔眼角膜缘干细胞缺乏的动物模型。将实验兔随机分为2组：中度组和重度组,每组22只,每只兔均选右眼建模、手术。用内径12mm,外径18mm的环形滤纸片浸透1mol/L NaOH,覆盖于实验兔角膜缘处,重度组接触1min,中度组接触30s。然后用0.9%氯化钠注射液冲洗角膜表面和结膜囊5分钟。烧伤后每天观察兔角膜变化,并于碱烧伤后稳定期对角膜新生血管程度和炎症程度进行评分。用HE染色方法观察比较正常兔角膜和碱烧伤后稳定期兔角膜的组织病理学差异；用免疫组织化学方法检测正常组和碱烧伤后稳定期时兔角膜中p63、CD68的表达情况；用Real time-PCR方法检测正常组和碱烧伤后稳定期时兔角膜中干细胞相关因子ANp63α、ABCG2、CK3,及炎症因子IL-1β、IL-6、IL-4、IL-10、IL-13、TNF-α、MCP-1、iNOS、TGF-β、 VEGF的表达量。
2、以羊膜为载体人角膜缘上皮细胞体外培养
角膜来自山东省眼科研究所眼库,取用角膜移植后剩余的角巩膜环,将角膜上皮细胞消化成单细胞后接种于去上皮羊膜上,每天观察细胞生长情况,待细胞铺满羊膜形成复层时进行移植手术
3、以羊膜为载体培养的人角膜缘上皮细胞移植术
取碱烧伤后处于稳定期的兔角膜进行手术。术中剪开全周球结膜,剥除角膜表而覆盖的血管膜,将培养有人角膜缘上皮细胞的羊膜覆盖于角膜表面,将羊膜于角膜缘处缝合5～8针固定于球结膜和巩膜之间。手术中注意保护细胞避免受到损伤,术毕行睑裂缝合避免角膜过度暴露。
4、手术后的临床疗效评价和供体细胞的存活状态检测
分别于术后即刻,术后1天、3天、7天、14天、21天和28天时用裂隙灯显微镜观察术后兔角膜变化,对角膜新生血管化程度和炎症程度进行评分,并利用裂隙灯照相系统进行图像记录。在上述各时间点取兔角膜进行HE染色,并用免疫组化方法检测供体和受体中p63、Ki-67的表达情况。用RT-PCR方法检测术前及术后各时间点供体和受体细胞中干细胞相关因子ANp63α、Ki-67、ABCG2、C/EBPA、CK3的表达量。
5、手术后眼表微环境中炎症因子表达情况检测
分别于手术后即刻、术后1天、3天、7天、14天、28天时取兔角膜,用免疫组化方法检测CD68的表达情况,用Real time-PCR方法检测炎症因子IL-1β、IL-6、IL-4、IL-10、IL-13、TNF-α、MCP-1、iNOS、TGF-β、VEGF、CD4、CD8的表达量。
结果
1、碱烧伤后兔角膜的病变特征
碱烧伤后烧伤部位的角膜即呈瓷白色混浊,碱烧伤后1周内为急性期,球结膜缺血,角膜水肿,角膜上皮持续缺损,碱烧伤后第2-3周进入损伤和修复共存期。中度碱烧伤组在烧伤后4-8周时角膜新生血管化,炎症反应趋于稳定；重度碱烧伤组在伤后8-12周时才能进入稳定期。中度碱烧伤组有3眼评分未达标而排除研究,重度碱烧伤组3眼角膜穿孔排除研究。根据评分标准,中度组和重度组的总分平均值分别为5.7±1.2分和8.8±1.1分,两组差别有统计学意义(P=0.000)。HE染色可见碱烧伤后稳定期时角膜表面覆盖扁平的鳞状上皮细胞,基质内见大量管径粗大的新生血管,可达深基质层。免疫组织化学检测可见大量CD68阳性的炎症细胞浸润,角膜中p63表达阴性。Real time-PCR结果显示碱烧伤组ANp63α、ABCG2、CK3的表达显著低于正常对照组,其中重度碱烧伤组降低较中度碱烧伤组降低更为显著(P<0.05)。碱烧伤后稳定期角膜中炎症因子IL-1β、 IL-6、IL-4、IL-10、IL-13、TNF-α、 iNOS、MCP-1、TGF-β、VEGF的表达显著高于正常对照组。
2、以羊膜为载体培养的兔角膜缘上皮细胞移植术后的临床疗效
中度碱烧伤组在术后1-28天时均可见到角膜上皮完整,未见新生血管再次生长。重度碱烧伤组在术后1天时可见到角膜上皮片状缺损,术后3天时角膜上皮修复完整,术后14天和21天时再次出现角膜上皮片状缺损,自术后14天时可见新生学管再次长入角膜,甚至比术前更为严重。HE染色可见术后7天内羊膜与角膜贴附不紧密,术后14天时羊膜吸收,但此时角膜上皮细胞与角膜基质仍有分离现象。中度碱烧伤组术后21天和28天时角膜上皮完整,角膜基质内未见新生血管管腔；重度碱烧伤组可见角膜上皮缺损,或角膜上皮不规则增生,呈鳞状上皮化,角膜基质内大量炎症细胞浸涧,并可见到新生血管管腔。
3、手术后供体细胞的存活状态
免疫组织化学检测在中度碱烧伤组术后28天内均可见供体p63和Ki-67表达。重度碱烧伤组术后7天内可见p63表达,偶见Ki-67阳性细胞,RT-PCR结果显示两组供体细胞中ANp63α的农达在手术后即刻降低约40%,术后3天时显著降低,而ABCG2在术后28天内均有表达但呈逐渐下降趋势。中度碱烧伤组Ki-67的表达量在术后7天内较术前升高,但术后14天时即显著降低至消失；C/EBPA和CK3的表达量在术后即刻较术前降低,但术后1天时有所上调,术后3天、7天时呈显著下降,至术后14天后几乎检测不到其表达。重度碱烧伤组Ki-67、C/EBP△、CK3的表达在术后3天时即显著降低,术后7天时表达量极低甚至消失。
4、手术后眼表炎症微环境中炎症因子的表达
HE染色和免疫组织化学结果：术后1天和3天时在羊膜下和角膜基质中可见分叶清晰的中性粒细胞浸润和CD68阳性的巨噬细胞浸润,术后7天时较少见到分叶清晰的中性粒细胞,但CD68阳性细胞仍较多。中度碱烧伤组术后14天后CD68阳性细胞逐渐减少,而重度碱烧伤组CD68阳性细胞浸润可持续至术后28天。
Real-time PCR结果：炎症因子IL-1β、IL-6、MCP-1、IL-13、IL-10的表达在术后即刻即显著上调,术后3天时显著下调,术后7天和14天再次上调,随后逐渐下降。大部分炎症因子在术后出现两个高峰,即术后即刻和术后7天时。CD4和CD8的表达在术后1天、3天均处在极低水平,术后7天时骤然上调,此后下降。
5、手术后受体中干细胞相关因子的表达
中度碱烧伤组ANp63α、ABCG2、CK3、Ki-67的表达在术后较术前呈上调趋势,在术后21天时较为显著。重度碱烧伤组ANp63α、ABCG2、CK3、Ki-67在术后的表达量没有超过术前,在术后早期如1天、3天、7天时甚至较术前降低。
结论
1、角膜缘环形碱烧伤是建立角膜缘干细胞缺乏模型的有效方法,在碱烧伤后稳定期角膜内仍有大量巨噬细胞浸润和多种炎症因子高表达,中度碱烧伤角膜缘内可能残存一些可恢复功能的干细胞。
2、移植的供体细胞在术后7天内可维持其增殖、分化和自我更新能力,随后细胞的各项功能均下降至丧失。
3、术后早期的炎症反应和免疫排斥反应是影响移植的供体细胞功能的关键因素,巨噬细胞可能在其中发挥了主要作用。
4、有足够的供体细胞长期存活并发挥正常功能是术后维持稳定眼表的一个必要条件,而受体自身残存足够的可恢复功能的角膜缘干细胞是术后维持稳定眼表的另一个重要条件。
Purpose:To investigate the survival state of the donor cells after ex vivo cultured limbal epithelial transplantation and analyze the possible influencing factors.
Methods:Rabbit limbal stem cell deficiency mode was induced by alkali burn, and ex vivo cultured limbal epithelial transplantation was performed at stable stage after alkali burn. Clinical manifestations were observed by slit lamp microscope at immediate postopereative, and1day,3day,7day,14day,21day,28day postoperative. HE stain was performed at preoperation and each time point, and the expression of p63, Ki-67, CD68in donor and host tissue were detected by immunohistochemistry. RT-PCR was used to detect the expression of ANp63α、 Ki-67、 ABCG2、 C/EBPA、 CK3and inflammatory factors IL-lβ、IL-6、 IL-4、 IL-10、 IL-13、 TNF-α、 MCP-1、TGF-β、 VEGF、CD4、CD8at each time point.
Results:In moderate group, the corneal epithelium is integral and without neovascularization within1month. But in severe group, corneal epithelial defected repeatedly and neovascularization was occurred at14days postoperation. HE stain and immunohistochemistry shows that p63and Ki-67were expressed within28days in moderate group, but gradually decreased. P63could be detected within7days in severe group, and Ki-67was rarely detected. A large number of CD68+macrophage infiltrated in preoperative corneal strom in both group. Neutrophils infiltrated in amniotic was detected at1day and3days postoperation and significantly reduced at7days postoperation. Macrophage consistently elevated in severe group, but gradually reduced14days later in moderate group. RT-PCR results show that the expression of ANp63a in donor cells was reduced about40%immediately postopereative, and significantly reduced at3days postopereative. The expression of ABCG2could be detected but gradually reduced within28days. In moderate group, the expression of Ki-67, C/EBPA, CK3were significantly reduced or even disappear at14days postoperative. But in severe group, the expression of Ki-67, C/EBP△, CK3were significantly reduced at3days postoperative, and at7days postoperative we can only detect very a low expression level or even disappear of these factors. Most inflammatory factors appear two peaks postoperative. The first peak appear immediately postopereative, was early inflammation mediated by macrophages. The expression of CD4was significantly increased at7days postopereative, and the second peak appears at this time. So the possible reason of this peak was immune rejection induced by CD4+cells. The expression of host△Np63a, ABCG2, CK3was increased compared with the preoperative in moderate group, but in severe group, it does not change or even decreased.
Conclusions:The donor cells could be exist within1month after ex vivo cultured limbal epithelial transplantation, but its proliferation, differentiation, and self-renewal capacity could significantly reduce or loss within14days under the hit of mechanical injury during the surgery, early inflammatory response postoperative and immune rejection. Macrophages may play an important role in reduce the early inflammatory and immune rejection. Functional recovery of the host stem cells maybe an important factor to maintain a long-term stable ocular surface.
体外培养的异体角膜缘上皮细胞移植术是治疗角膜缘干细胞缺乏的有效方案,在临床中我们观察到术后有些患者会发生再次角膜新生血管化和角膜上皮反复缺损,考虑其原因可能为手术后供体细胞不能很好的存活并发挥功能。因此,本研究的目的是通过动物模型模拟该手术及术后的临床过程,探讨手术后供体细胞的存活状态和其可能的影响因素,并进一步探讨手术后可维持眼表稳定的可行措施。
方法
1、动物模型的建立和评价
采用4周龄雌性新西兰大白兔45只,建立碱烧伤致兔眼角膜缘干细胞缺乏的动物模型。将实验兔随机分为2组：中度组和重度组,每组22只,每只兔均选右眼建模、手术。用内径12mm,外径18mm的环形滤纸片浸透1mol/L NaOH,覆盖于实验兔角膜缘处,重度组接触1min,中度组接触30s。然后用0.9%氯化钠注射液冲洗角膜表面和结膜囊5分钟。烧伤后每天观察兔角膜变化,并于碱烧伤后稳定期对角膜新生血管程度和炎症程度进行评分。用HE染色方法观察比较正常兔角膜和碱烧伤后稳定期兔角膜的组织病理学差异；用免疫组织化学方法检测正常组和碱烧伤后稳定期时兔角膜中p63、CD68的表达情况；用Real time-PCR方法检测正常组和碱烧伤后稳定期时兔角膜中干细胞相关因子ANp63α、ABCG2、CK3,及炎症因子IL-1β、IL-6、IL-4、IL-10、IL-13、TNF-α、MCP-1、iNOS、TGF-β、 VEGF的表达量。
2、以羊膜为载体人角膜缘上皮细胞体外培养
角膜来自山东省眼科研究所眼库,取用角膜移植后剩余的角巩膜环,将角膜上皮细胞消化成单细胞后接种于去上皮羊膜上,每天观察细胞生长情况,待细胞铺满羊膜形成复层时进行移植手术
3、以羊膜为载体培养的人角膜缘上皮细胞移植术
取碱烧伤后处于稳定期的兔角膜进行手术。术中剪开全周球结膜,剥除角膜表而覆盖的血管膜,将培养有人角膜缘上皮细胞的羊膜覆盖于角膜表面,将羊膜于角膜缘处缝合5～8针固定于球结膜和巩膜之间。手术中注意保护细胞避免受到损伤,术毕行睑裂缝合避免角膜过度暴露。
4、手术后的临床疗效评价和供体细胞的存活状态检测
分别于术后即刻,术后1天、3天、7天、14天、21天和28天时用裂隙灯显微镜观察术后兔角膜变化,对角膜新生血管化程度和炎症程度进行评分,并利用裂隙灯照相系统进行图像记录。在上述各时间点取兔角膜进行HE染色,并用免疫组化方法检测供体和受体中p63、Ki-67的表达情况。用RT-PCR方法检测术前及术后各时间点供体和受体细胞中干细胞相关因子ANp63α、Ki-67、ABCG2、C/EBPA、CK3的表达量。
5、手术后眼表微环境中炎症因子表达情况检测
分别于手术后即刻、术后1天、3天、7天、14天、28天时取兔角膜,用免疫组化方法检测CD68的表达情况,用Real time-PCR方法检测炎症因子IL-1β、IL-6、IL-4、IL-10、IL-13、TNF-α、MCP-1、iNOS、TGF-β、VEGF、CD4、CD8的表达量。
结果
1、碱烧伤后兔角膜的病变特征
碱烧伤后烧伤部位的角膜即呈瓷白色混浊,碱烧伤后1周内为急性期,球结膜缺血,角膜水肿,角膜上皮持续缺损,碱烧伤后第2-3周进入损伤和修复共存期。中度碱烧伤组在烧伤后4-8周时角膜新生血管化,炎症反应趋于稳定；重度碱烧伤组在伤后8-12周时才能进入稳定期。中度碱烧伤组有3眼评分未达标而排除研究,重度碱烧伤组3眼角膜穿孔排除研究。根据评分标准,中度组和重度组的总分平均值分别为5.7±1.2分和8.8±1.1分,两组差别有统计学意义(P=0.000)。HE染色可见碱烧伤后稳定期时角膜表面覆盖扁平的鳞状上皮细胞,基质内见大量管径粗大的新生血管,可达深基质层。免疫组织化学检测可见大量CD68阳性的炎症细胞浸润,角膜中p63表达阴性。Real time-PCR结果显示碱烧伤组ANp63α、ABCG2、CK3的表达显著低于正常对照组,其中重度碱烧伤组降低较中度碱烧伤组降低更为显著(P<0.05)。碱烧伤后稳定期角膜中炎症因子IL-1β、 IL-6、IL-4、IL-10、IL-13、TNF-α、 iNOS、MCP-1、TGF-β、VEGF的表达显著高于正常对照组。
2、以羊膜为载体培养的兔角膜缘上皮细胞移植术后的临床疗效
中度碱烧伤组在术后1-28天时均可见到角膜上皮完整,未见新生血管再次生长。重度碱烧伤组在术后1天时可见到角膜上皮片状缺损,术后3天时角膜上皮修复完整,术后14天和21天时再次出现角膜上皮片状缺损,自术后14天时可见新生学管再次长入角膜,甚至比术前更为严重。HE染色可见术后7天内羊膜与角膜贴附不紧密,术后14天时羊膜吸收,但此时角膜上皮细胞与角膜基质仍有分离现象。中度碱烧伤组术后21天和28天时角膜上皮完整,角膜基质内未见新生血管管腔；重度碱烧伤组可见角膜上皮缺损,或角膜上皮不规则增生,呈鳞状上皮化,角膜基质内大量炎症细胞浸涧,并可见到新生血管管腔。
3、手术后供体细胞的存活状态
免疫组织化学检测在中度碱烧伤组术后28天内均可见供体p63和Ki-67表达。重度碱烧伤组术后7天内可见p63表达,偶见Ki-67阳性细胞,RT-PCR结果显示两组供体细胞中ANp63α的农达在手术后即刻降低约40%,术后3天时显著降低,而ABCG2在术后28天内均有表达但呈逐渐下降趋势。中度碱烧伤组Ki-67的表达量在术后7天内较术前升高,但术后14天时即显著降低至消失；C/EBPA和CK3的表达量在术后即刻较术前降低,但术后1天时有所上调,术后3天、7天时呈显著下降,至术后14天后几乎检测不到其表达。重度碱烧伤组Ki-67、C/EBP△、CK3的表达在术后3天时即显著降低,术后7天时表达量极低甚至消失。
4、手术后眼表炎症微环境中炎症因子的表达
HE染色和免疫组织化学结果：术后1天和3天时在羊膜下和角膜基质中可见分叶清晰的中性粒细胞浸润和CD68阳性的巨噬细胞浸润,术后7天时较少见到分叶清晰的中性粒细胞,但CD68阳性细胞仍较多。中度碱烧伤组术后14天后CD68阳性细胞逐渐减少,而重度碱烧伤组CD68阳性细胞浸润可持续至术后28天。
Real-time PCR结果：炎症因子IL-1β、IL-6、MCP-1、IL-13、IL-10的表达在术后即刻即显著上调,术后3天时显著下调,术后7天和14天再次上调,随后逐渐下降。大部分炎症因子在术后出现两个高峰,即术后即刻和术后7天时。CD4和CD8的表达在术后1天、3天均处在极低水平,术后7天时骤然上调,此后下降。
5、手术后受体中干细胞相关因子的表达
中度碱烧伤组ANp63α、ABCG2、CK3、Ki-67的表达在术后较术前呈上调趋势,在术后21天时较为显著。重度碱烧伤组ANp63α、ABCG2、CK3、Ki-67在术后的表达量没有超过术前,在术后早期如1天、3天、7天时甚至较术前降低。
结论
1、角膜缘环形碱烧伤是建立角膜缘干细胞缺乏模型的有效方法,在碱烧伤后稳定期角膜内仍有大量巨噬细胞浸润和多种炎症因子高表达,中度碱烧伤角膜缘内可能残存一些可恢复功能的干细胞。
2、移植的供体细胞在术后7天内可维持其增殖、分化和自我更新能力,随后细胞的各项功能均下降至丧失。
3、术后早期的炎症反应和免疫排斥反应是影响移植的供体细胞功能的关键因素,巨噬细胞可能在其中发挥了主要作用。
4、有足够的供体细胞长期存活并发挥正常功能是术后维持稳定眼表的一个必要条件,而受体自身残存足够的可恢复功能的角膜缘干细胞是术后维持稳定眼表的另一个重要条件。
Purpose:To investigate the survival state of the donor cells after ex vivo cultured limbal epithelial transplantation and analyze the possible influencing factors.
Methods:Rabbit limbal stem cell deficiency mode was induced by alkali burn, and ex vivo cultured limbal epithelial transplantation was performed at stable stage after alkali burn. Clinical manifestations were observed by slit lamp microscope at immediate postopereative, and1day,3day,7day,14day,21day,28day postoperative. HE stain was performed at preoperation and each time point, and the expression of p63, Ki-67, CD68in donor and host tissue were detected by immunohistochemistry. RT-PCR was used to detect the expression of ANp63α、 Ki-67、 ABCG2、 C/EBPA、 CK3and inflammatory factors IL-lβ、IL-6、 IL-4、 IL-10、 IL-13、 TNF-α、 MCP-1、TGF-β、 VEGF、CD4、CD8at each time point.
Results:In moderate group, the corneal epithelium is integral and without neovascularization within1month. But in severe group, corneal epithelial defected repeatedly and neovascularization was occurred at14days postoperation. HE stain and immunohistochemistry shows that p63and Ki-67were expressed within28days in moderate group, but gradually decreased. P63could be detected within7days in severe group, and Ki-67was rarely detected. A large number of CD68+macrophage infiltrated in preoperative corneal strom in both group. Neutrophils infiltrated in amniotic was detected at1day and3days postoperation and significantly reduced at7days postoperation. Macrophage consistently elevated in severe group, but gradually reduced14days later in moderate group. RT-PCR results show that the expression of ANp63a in donor cells was reduced about40%immediately postopereative, and significantly reduced at3days postopereative. The expression of ABCG2could be detected but gradually reduced within28days. In moderate group, the expression of Ki-67, C/EBPA, CK3were significantly reduced or even disappear at14days postoperative. But in severe group, the expression of Ki-67, C/EBP△, CK3were significantly reduced at3days postoperative, and at7days postoperative we can only detect very a low expression level or even disappear of these factors. Most inflammatory factors appear two peaks postoperative. The first peak appear immediately postopereative, was early inflammation mediated by macrophages. The expression of CD4was significantly increased at7days postopereative, and the second peak appears at this time. So the possible reason of this peak was immune rejection induced by CD4+cells. The expression of host△Np63a, ABCG2, CK3was increased compared with the preoperative in moderate group, but in severe group, it does not change or even decreased.
Conclusions:The donor cells could be exist within1month after ex vivo cultured limbal epithelial transplantation, but its proliferation, differentiation, and self-renewal capacity could significantly reduce or loss within14days under the hit of mechanical injury during the surgery, early inflammatory response postoperative and immune rejection. Macrophages may play an important role in reduce the early inflammatory and immune rejection. Functional recovery of the host stem cells maybe an important factor to maintain a long-term stable ocular surface.
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