角膜内皮细胞生理和再生特性研究进展

角膜内皮细胞膜上的Na -K -ATP酶、水通道蛋白-1和各类离子通道相辅相成,发挥离子和水液的跨膜转运功能,共同维持角膜的透明状态.影响角膜内皮细胞生理功能的因素,主要是通过影响Na -K -ATP酶、离子通道和水通道蛋白而发挥作用的.人类角膜内皮细胞再生能力具有年龄差异、区域差异、在体和离体差异,以及是否存在干细胞还存在争议等特点.成年人角膜内皮细胞失去有丝分裂能力,主要与细胞接触抑制、TGF-β2和p27kip1有密切关系.从分子水平和基因层面调控角膜内皮细胞的生理功能和再生能力,将为角膜病的防治提供新的思路.     

超声乳化白内障手术后患者角膜内皮细胞损伤的研究

目的:探讨超声乳化白内障手术患者角膜内皮细胞的损伤情况。方法:收集我院确诊为白内障的患者121例,随机分配为微切口组与常规切口组。常规切口组采用3.0 mm切口超声乳化白内障手术方案,微切口组采用1.8 mm小切口超声乳化白内障手术。手术前、手术后1日、7日、1个月、3个月监测患者角膜内皮细胞密度、六角形细胞比例、角膜内皮细胞变异系数及中央角膜厚度。结果:与治疗前相比,两组患者手术后1日、7日、1个月、3个月的角膜内皮细胞密度、六角形细胞比例及角膜内皮细胞变异系数均较治疗前显著降低(P0.05);手术后1日、7日时,两组患者的中央角膜厚度均较手术前明显变薄,有统计学差异(P0.05);手术后1个月、3个月,两组患者的中央角膜厚度均呈降低趋势,最终与手术前相似。微切口组患者不同时点六角形细胞比例与同期常规切口组比较均显著升高,角膜内皮细胞变异系数与同期常规切口组比较均明显降低有统计学差异(P0.05)。两组患者手术前后不同时间点角膜内皮细胞密度、中央角膜厚度组间比较均无统计学差异(P0.05)。结论:超声乳化白内障手术后患者角膜内皮细胞损伤与手术切口有相关性,缩小手术面积的小切口手术使术后修复增快,安全有效,适宜临床推广。     

角膜内皮细胞的检测、受损因素及治疗新进展

摘要：角膜内皮细胞(corneal endothelial cells,CECs)通过其屏障作用及离子泵功能维持角膜透明,但年龄、疾病及创伤等因素会使其功能有所降低,导致角膜失代偿、角膜水肿、大泡性角膜病变,甚至视力的丧失。因此,CECs可谓是角膜的"生命"。近年来,CECs的检测方法逐渐新增,并且不同检测指标具有不同意义;导致CECs受损的因素也有所增加,同时CECs损伤后的治疗也逐渐成为近年来的研究热点。早期发现CECs受损、明确病因并进行干预或治疗会减少CECs的损伤,对指导临床工作有重要的意义。本文主要对CECs检测方法及指标、损伤因素及其治疗的最新进展进行了综述。     

小鼠角膜上皮祖细胞系TKE2的表型研究

目的：观察小鼠角膜上皮祖细胞系TKE2在扩增以及分化状态下的角蛋白及干细胞标志物的表达情况。方法小鼠角膜上皮祖细胞系TKE2在无血清培养基Keratinocyte-SFM （KSFM）以及含10﹪胎牛血清（FBS）的DMEM培养基中培养,约70﹪融合时进行角蛋白10、12、14、15、16（K10、K12、K14、K15、K16）以及Connexin43、ABCG2的免疫荧光染色,以及Ki67、P63、PCNA的免疫细胞化学染色。结果无血清培养状态下的TKE2细胞呈克隆样生长,克隆内所有细胞呈ABCG2、K14、Ki67、PCNA以及P63阳性,K15阳性细胞散在分布,K16阳性细胞呈片状分布于克隆中央区,K10、K12以及Connexin43染色为阴性。在含有10﹪胎牛血清的DMEM中培养2 d后,细胞明显增大, ABCG2、K15、P63、Ki67以及PCNA转为阴性,克隆内只有少量细胞呈K16、K14阳性染色, K10、K12、Connexin43仍为阴性。结论 TKE2细胞具有角膜上皮干细胞特性,可以作为角膜缘上皮干细胞表型维持和分化诱导研究的良好工具。     

不同缝合方式对大鼠角膜穿通伤后内皮细胞影响的研究

目的:通过手术缝合治疗大鼠角膜穿通伤,探索全层缝合、深板层缝合、及不缝合对角膜内皮细胞的影响.方法:建立大鼠角膜穿通伤模型,同一手术者对角膜切口进行全层、深板层、及不缝合操作.在裂隙灯下动态观察角膜创伤愈合情况;对不同时间点愈合角膜行内皮细胞台盼蓝-茜素红联合活细胞染色及HE染色,观察内皮细胞损伤、修复及白细胞浸润情况.结果:无论是全层缝合还是板层缝合以及不缝合组角膜内皮细胞均损伤明显.但从第1天观察至1月,三组损伤区面积大小无明显差别.结论:全层和深板层缝合及未缝合组可直接造成角膜内皮细胞受损,继发性炎症反应损伤后角膜内皮细胞的损害;伤口周围1.5 mm处角膜内皮几乎损失殆尽,内皮细胞受损可使角膜损伤区水肿迁延不愈,最终形成瘢痕愈合,所以角膜内皮损伤及最终愈合程度三组间无明显差异.     

兔角膜缘干细胞的研究进展

目前,角膜移植是临床上治疗角膜疾患的最有效途径,但供体角膜非常有限。新近兴起的干细胞技术,为组织工程角膜的研制和应用提供了契机。对于以角膜缘干细胞缺乏或功能障碍为特征的疾病也有治疗效果。采取角膜缘干细胞移植应是一种合理有效的治疗手段。本文主要介绍了兔角膜缘干细胞的体外分离、培养、鉴定及一些生长因子对其增殖的影响。     

小梁切除术中应用丝裂霉素C 对角膜内皮细胞的影响

目的:观察小梁切除术中应用丝裂霉素C(MMC)对角膜内皮细胞的影响。方法:收集2010年9月2011年5月在我院行小梁切除术的青光眼患者60例(78眼),随机分为术中应用丝裂霉素C的36例(46眼)患者为A组,术中不用丝裂霉素C的24例(32眼)为B组,分别观察术前、术后1个月和术后3个月两组眼压(IOP)、角膜内皮细胞的密度(CD)、平均细胞面积(AVG)及细胞面积变异系数(CV),分析其数量的改变及两组间的差异。结果:A组术前眼压为(35.4±13.7)mmHg,B组术前眼压为(32.5±13.5)mmHg差异无统计学意义(P>0.05),A组术后1个月及术后3个月眼压分别为(15.7±3.7)mmHg、(17.0±3.2)mmHg,均低于B组的(19.4±3.7)mmHg、(20.2±2.1)mmHg,差异有统计学意义(P<0.05)。A组术前、术后1个月及术后3个月角膜内皮细胞密度分别为(2475±484)个/mm2、(2199±373)个/mm2、(2164±332)个/mm2;平均细胞面积分别为(431.4±67.6)μm2、(480.6±66.8)μm2、(463.8±46.2)μm2;细胞面积变异系数分别为(31.1±7.4)%、(34.4±6.3)%、(31.2±7.5)%;术后1个月及术后3个月各参数与术前比较,差异均有统计学意义(P<0.05)。B组术前、术后1个月及术后3个月角膜内皮细胞密度分别为(2342±94)个/mm2、(2185±215)个/mm2、(2074±218)个/mm2;平均细胞面积分别为(453.9±94.8)μm2、(516.3±100.8)μm2、(499.81±106.4)μm2;细胞面积变异系数分别为(30.2±3.0)%、(32.7±2.9)%、(31.4±4.3)%;除术后3个月角膜内皮细胞与术前比较有意义(P<0.05)外,余参数术后1个月及术后3个月与术前比较差异均无统计学意义(P>0.05)。术后1个月A组的角膜内皮细胞丢失率为10.4%高于B组的6.1%,差异有统计学意义(P<0.05);术后3个月A组的角膜内皮细胞丢失率为11.1%高于B组的10.0%,差异无统计学意义(P>0.05)。结论:小梁切除术中用丝裂霉素C的降压效果比不用丝裂霉素C的效果好,但短期内前者角膜内皮细胞的丢失率高于后者。     

小梁切除术中应用丝裂霉素C对角膜内皮细胞的影响

目的：观察小梁切除术中应用丝裂霉素C（MMC）对角膜内皮细胞的影响。方法：收集2010年9月2011年5月在我院行小梁切除术的青光眼患者60例（78眼）,随机分为术中应用丝裂霉素c的36例（46眼）患者为A组,术中不用丝裂霉素c的24例（32眼）为B组。分别观察术前、术后1个月和术后3个月两组眼压（10P）、角膜内皮细胞的密度（co）、平均细胞面积（AVG）及细胞面积变异系数（cv）,分析其数量的改变及两组间的差异。结果：A组术前眼压为（35．4±13．7）mmHg,B组术前眼压为（32．5±13．5）mmHg差异无统计学意义（P〉0．05）,A组术后1个月及术后3个月眼压分别为（15．7±3．7）mmHg、（17．0±3．2）mmHg,均低于B组的（19．4±3．7）mmHg、（20．2±2．1）mmHg,差异有统计学意义（P〈0．05）。A组术前、术后1个月及术后3个月角膜内皮细胞密度分别为（2475±484）个／mm2、（2199±373）个／mm2、（2164±332）个／mm2;平均细胞面积分别为（431．4±67．6）μm2、（480．6±66．8）μm2、（463．8±46．2）μm2;细胞面积变异系数分别为（31．1±7．4）％、（34．4±6．3）％、（31．2±7．5）％;术后1个月及术后3个月各参数与术前比较,差异均有统计学意义（P〈0．05）。B组术前、术后1个月及术后3个月角膜内皮细胞密度分别为（2342±94）个／mm2、（2185＋215）个／mm2、（2074218）个／mm2;平均细胞面积分别为（453．9土94．8）μm2、（516.3±100．8）μm2、（499．81＋106．4）μm2;细胞面积变异系数分别为（30．2土3．0）％、（32．7±2．9）％、（31．4±4．3）％;除术后3个月角膜内皮细胞与术前比较有意义（P〈0．05）外,余参数术后1个月及术后3个月与术前比较差异均无统计学意义（P〉0．05）。术后1个月A组的角膜内皮细胞丢失率为10．4％高于B组的6．1％,差异有统计学意义（P〈0．05）;术后3个月A组的角膜内皮细胞丢失率为11．1％高于B组的10．0％,差异无统计学意义（P〉0．05）。结论：小梁切除术中用丝裂霉素C的降压效果比不用丝裂霉素C的效果好,但短期内前者角膜内皮细胞的丢失率高于后者。     

白内障超声乳化术对糖尿病患者角膜内皮细胞和中央角膜厚度影响

目的:评估糖尿病患者和非糖尿病患者在行白内障超声乳化手术后,角膜内皮细胞和中央角膜厚度变化。方法:观察于我院行白内障超声乳化吸出联合人工晶状体植入术的年龄相关性白内障患者各100例(100眼),于术前1天、术后1周、1个月、3个月、6个月和1年随访记录其角膜内皮细胞密度(endothelial cell density, ECD)、六角形细胞百分比(percentage of hexagonal cells,PHC)、变异系数(coefficient of variation, CV)和中央角膜厚度(central corneal thickness, CCT)等指标,并对结果进行统计学分析。结果:非糖尿病组在术前各指标无明显差异(P0.05),术后1年ECD, PHC在两组均下降,CV升高(P0.05),CCT出现明显波动,糖尿病组在术后一周达到峰值。ECD,CV和PHC在术后各时间点出现明显的组间差异(P0.05)。结论:白内障超声乳化术后远期内,糖尿病组的角膜内细胞和中央角膜厚度较非糖尿病组发生显著变化。     

框架眼镜联合角膜塑形镜矫正青少年高度近视的临床疗效及对角膜内皮细胞和泪膜的影响

目的:探讨框架眼镜联合角膜塑形镜矫正青少年高度近视的临床疗效及对角膜内皮细胞和泪膜的影响.方法:选取2018年1月～2019年3月期间我院收治的青少年高度近视患者98例(181眼),根据信封抽签法分为对照组49例92眼(框架眼镜配戴治疗)和观察组49例89眼(对照组基础上联合角膜塑形镜治疗).对比两组裸眼视力、屈光度、...     

角膜上皮细胞代谢的研究进展

角膜上皮层位于角膜表面,外邻泪膜,内与角膜前弹力层相连。角膜上皮细胞代谢所需营养及氧分主要通过泪膜、房水和角膜缘毛细血管运送。正常的角膜上皮细胞代谢是维持角膜上皮细胞正常增殖与分化状态的关键。角膜上皮细胞代谢异常可导致上皮损伤或变性,是多种角膜疾病的病理基础。本文就近年来关于角膜上皮细胞代谢相关的组织结构、营养来源、细胞增殖分化以及相关疾病的研究进展进行综述。     

Effects of shear stress on differentiation of stem cells into endothelial cells

Stem cell transplantation is an appealing potential therapy for vascular diseases and an indispensable key step in vascular tissue engineering. Substantial effort has been made to differentiate stem cells toward vascular cell phenotypes, including endothelial cells (ECs) and smooth muscle cells. The microenvironment of vascular cells not only contains biochemical factors that influence differentiation but also exerts hemodynamic forces, such as shear stress and cyclic strain. More recently, studies have shown that shear stress can influence the differentiation of stem cells toward ECs. A deep understanding of the responses and underlying mechanisms involved in this process is essential for clinical translation. This review highlights current data supporting the role of shear stress in stem cell differentiation into ECs. Potential mechanisms and signaling cascades for transducing shear stress into a biological signal are proposed. Further study of stem cell responses to shear stress will be necessary to apply stem cells for pharmacological applications and cardiovascular implants in the realm of regenerative medicine.     

Caution regarding the combined effects of extracellular matrices and nutrient media on cultured endothelial cells

To study the influence of smooth muscle cells (SMC) on endothelial cells (EC), different co-culture designs are available, including EC seeding on SMC extracellular matrix (ECM). We explored human umbilical vein endothelial cell (HUVEC) adhesion and proliferation on either in situ or coated ECM, elaborated by HUVECs or human arterial smooth muscle cells (HUASMCs), in the presence of different nutrient media containing varying amounts of fetal calf serum. Coating wells with HUVEC or HUASMC ECMs did not improve HUVEC adhesion 1 h after cell seeding, compared with uncoated wells. HUVEC adhesion on in situ HUVEC-ECM and HUASMC-ECM was significantly increased compared with uncoated wells. The substratum upon which cells are maintained was found to play a crucial role, in conjunction with the medium to which HUVECs are exposed for their proliferative response. These results stress the importance of selecting media in relation to the particular substratum, in order to avoid misinterpretation of data.     

P2Y11 impairs cell proliferation by induction of cell cycle arrest and sensitizes endothelial cells to cisplatin-induced cell death

Extracellular ATP mediates a wide range of physiological effects, including cell proliferation, differentiation, maturation, and migration. However, the effect of ATP on cell proliferation has been contradictory, and the mechanism is not fully understood. In the current study, we found that extracellular ATP significantly inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs). Treatment with ATP did not induce cell apoptosis but instead induced cell cycle arrest in S phase. ATP induced the phosphorylation of ERK1/2, but the ERK inhibitors, U0126 and PD9809, did not regulate the inhibition of cell proliferation induced by ATP. However, ATP-induced inhibition of cell proliferation was blocked by suramin, a nonspecific antagonist of the P2Y receptors, and endothelial cells expressed P2Y11, a P2Y receptor that specifically binds ATP. Moreover, the down-regulation of P2Y11 by RNA interference not only reversed the inhibition of cell proliferation but also ameliorated cell cycle arrest in S phase. In addition, P2Y11 sensitized endothelial cells to cisplatin-induced cell death by down-regulation of the expression of Bcl-2. Taken together, these results suggest that extracellular ATP impairs cell proliferation by triggering signaling to induce cell cycle arrest and sensitizes cell to death via P2Y11 in endothelial cells.     

Visualizing the cell-cycle progression of endothelial cells in zebrafish

The formation of vascular structures requires precisely controlled proliferation of endothelial cells (ECs), which occurs through strict regulation of the cell cycle. However, the mechanism by which EC proliferation is coordinated during vascular formation remains largely unknown, since a method of analyzing cell-cycle progression of ECs in living animals has been lacking. Thus, we devised a novel system allowing the cell-cycle progression of ECs to be visualized in vivo. To achieve this aim, we generated a transgenic zebrafish line that expresses zFucci (zebrafish fluorescent ubiquitination-based cell cycle indicator) specifically in ECs (an EC-zFucci Tg line). We first assessed whether this system works by labeling the S phase ECs with EdU, then performing time-lapse imaging analyses and, finally, examining the effects of cell-cycle inhibitors. Employing the EC-zFucci Tg line, we analyzed the cell-cycle progression of ECs during vascular development in different regions and at different time points and found that ECs proliferate actively in the developing vasculature. The proliferation of ECs also contributes to the elongation of newly formed blood vessels. While ECs divide during elongation in intersegmental vessels, ECs proliferate in the primordial hindbrain channel to serve as an EC reservoir and migrate into basilar and central arteries, thereby contributing to new blood vessel formation. Furthermore, while EC proliferation is not essential for the formation of the basic framework structures of intersegmental and caudal vessels, it appears to be required for full maturation of these vessels. In addition, venous ECs mainly proliferate in the late stage of vascular development, whereas arterial ECs become quiescent at this stage. Thus, we anticipate that the EC-zFucci Tg line can serve as a tool for detailed studies of the proliferation of ECs in various forms of vascular development in vivo.     

Current progress in use of adipose derived stem cells in peripheral nerve regeneration

Unlike central nervous system neurons; those in the peripheral nervous system have the potential for full regeneration after injury. Following injury, recovery is controlled by schwann cells which replicate and modulate the subsequent immune response. The level of nerve recovery is strongly linked to the severity of the initial injury despite the significant advancements in imaging and surgical techniques. Multiple experimental model shave been used with varying successes to augment the natural regenerative processes which occur following nerve injury. Stem cell therapy in peripheral nerve injury may be an important future intervention to improve the best attainable clinical results. In particular adipose derived stem cells(ADSCs) are multipotent mesenchymal stem cells similar to bone marrow derived stem cells, which are thought to have neurotrophic properties and the ability to differentiate into multiple lineages. They are ubiquitous within adipose tissue; they can form many structures resembling the mature adult peripheral nervous system. Following early in vitro work; multiple small and large animal in vivo models have been used in conjunction with conduits, autografts and allografts to successfully bridge the peripheral nerve gap. Some of the ADSC related neuroprotective and regenerative properties have been elucidated however much work remains before a model can be used successfully in human peripheral nerve injury(PNI). This review aims to provide a detailed overview of progress made in the use of ADSC in PNI, with discussion on the role of a tissue engineered approach for PNI repair.     

Aspirin inhibits human coronary artery endothelial cell proliferation by upregulation of p53

Aspirin (acetylsalicylic acid, ASA) is effective in the primary and secondary prevention of vascular events. This effect is mediated in large part by platelet inhibition; however, non-platelet-mediated effects may also be relevant in the overall efficacy of ASA. We determined the effect of ASA on the synthesis of DNA and total proteins in cultured human coronary endothelial cells (HCAECs). Fourth generation HCAECs were cultured and treated with ASA and rate of synthesis of DNA and total proteins was determined by incorporation of [3H]thymidine and [3H]proline, respectively. ASA inhibited DNA synthesis by 50% at a concentration of 1mM and protein synthesis by 50% at a concentration of 2mM. The inhibitory effect of ASA was observed as early as 2h after treatment of HCAECs. The inhibition of DNA and protein synthesis could be reversed within 24h after removal of the drug from the culture medium. Indomethacin also inhibited DNA and protein synthesis. Western blot analysis revealed that the expression of p53 protein was increased after treatment of the cells with ASA. These observations indicate that ASA decreases endothelial cell proliferation through cell cycle arrest mediated by enhanced p53 expression. Arrest of endothelial proliferation and activation may be an important mechanism of the beneficial effect of ASA in acute coronary syndromes.