新鲜羊膜移植治疗眼部碱烧伤的临床观察

目的探讨新鲜羊膜移植对不同程度角膜碱烧伤的治疗效果及治疗时机的选择。方法角膜碱烧伤病人17例（17眼）,Ⅱ度7眼,Ⅲ度8眼,Ⅳ度2眼。均采用新鲜羊膜移植覆盖角膜、结膜治疗。观察术前及术后病人的视力、角膜、结膜、睑球粘连、角膜新生血管等情况。结果Ⅱ度7例（7眼）术后角膜透明,视力较术前均提高0.2～0.4;Ⅲ度8例（8眼）术后角膜见云翳和斑翳,视力较术前均提高0.1～0.3。上述15例均无睑球粘连及角膜新生血管长入。Ⅳ度2例（2眼）术后角膜溃疡面瘢痕化,有新生血管长入,视力无提高。结论新鲜羊膜移植治疗早期眼碱烧伤效果显著,术后视力恢复佳,可以减少瘢痕及新生血管发生,能很好地预防睑球粘连。     

Propolis and amnion reepithelialise second-degree burns in rats

Burns are serious consequences of trauma in terms of both imminent mortality and prolonged periods of morbidity. They are often accompanied by unsatisfactory cosmetic as well as functional and psychological outcomes. These complications emphasise the need for stronger efforts in achieving greater diversity and effectiveness in the treatment of skin burns. This study aimed to verify the effectiveness of gross and microscopic epidermal and dermal responses in the process of regenerative repair or healing of burns in rats that were treated either daily with 5% propolis ointment or by autologous amnion graft. Second-degree burns were inflicted in the neck region of female rats by contact with a hot metal (at 130 °C) for 5 s. Propolis treatment accelerated the process of tissue repair and led to decreased local inflammation, which indicates that treatment with propolis was successful in the initial period (7 days) and stimulated the production of collagen fibre (assessed by morphometry) in all the periods evaluated (14 and 21 days). Amnion treatment inhibited local inflammation (assessed macroscopically), stimulated local epithelial regeneration (assessed microscopically) and stimulated the production of collagen fibre (assessed by morphometry) in the days following burn. These treatments offer new therapeutic strategies for treating severe skin burns; these strategies may allow the minimisation of scar formation, a more rapid return of function and, ultimately, a better quality of life for burn patients.     

介入性宫内干预后绒毛膜羊膜分离的临床分析

目的：探讨介入性宫内干预后出现绒毛膜羊膜分离的临床特点及高危因素。方法：回顾性分析中山大学附属第一医院胎儿中心2001年7月—2010年6月行介入性宫内干预术后超声检查发现绒毛膜羊膜分离的病例,介入性干预包括羊水减量术213例、宫内输血28例、电凝脐带术28例。结果：269例宫内干预术后的病例中,共发现9例绒毛膜羊膜分离,羊水减量术后4例（4/213,1.88%）,宫内输血术后1例（1/28,3.6%）,电凝脐带术后4例（4/28,14.3%）。结论：电凝脐带术后绒毛膜羊膜分离发生率高,双胎输血综合征及羊水过多可能是绒毛膜羊膜分离发生的高危因素。     

不同冻存方法对羊膜超微结构和上皮细胞活性的影响

背景：羊膜冻存方法众多,对羊膜超微结构和生物活性的影响不一,目前尚无有效的冻存方法。 目的：比较不同冻存方法对羊膜超微结构和活性影响的研究,探寻更为理想的冻存方法。 方法：将新鲜羊膜采用深低温和玻璃化冻存法保存,分别于冻存后3,6个月复苏羊膜,以新鲜羊膜组织为对照组,比较羊膜的超微结构差异、羊膜上皮细胞离体氧分压和乳酸脱氢酶活性。 结果与结论：不同冻存方法保存的羊膜超微结构有明显改变,但玻璃化冻存对其超微结构的影响相对较小;与新鲜羊膜相比较,深低温冻存组3,6个月羊膜的乳酸脱氢酶灰度值和氧分压明显降低(P < 0.05);玻璃化冻存组6个月后的羊膜乳酸脱氢酶灰度值和氧分压明显降低(P < 0.05),而玻璃化冻存组3个月后的上述检测结果与新鲜羊膜相比差异无显著性意义(P > 0.05)。结果证实,羊膜的玻璃化冻存技术优于深低温冻存技术,不仅维持了羊膜的超微结构,而且保持了羊膜上皮细胞的功能和活性。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

羊膜上皮细胞损伤后在纤维蛋白支架及细胞生长因子作用下修复状况的研究

目的探讨人羊膜上皮细胞（HAEC）损伤后能否在体外构建的纤维蛋白支架上修复,以及表皮生长因子（EGF）、碱性成纤维细胞生长因子（bFGF）和转化生长因子β1（TGF-β1）对HAEC增殖的影响。方法采用环钻钻切培养板上的HAEC建立定量损伤模型,用制备的纤维蛋白块覆盖环钻钻切范围,分别加入不同浓度的培养液进行培养。其中加入EGF为EGF组、加入bFGF为bFGF组、加入TGF-β1为TGF-β组,不加任何细胞生长因子作为对照组。显微镜下观察各组HAEC生长移行情况,5-溴脱氧尿嘧啶掺入法检测HAEC的增殖效应。结果（1）各组HAEC均能向环钻缺损处移行并向内生长,EGF组、bFGF组的HAEC移行速度快,细胞数量多,对照组次之,TGF-β1组最少。（2）EGF组不同浓度的EGF（1．0、5．0、10．0、20．0、40．0、80．0及160．0ng／ml）培养下,HAEC细胞增殖率分别为17．8％、28．0％、35．3％、51．6％、34．1％、34．2％及26．0％,EGF组10～80ng／ml的HAEC增殖效应均明显大于对照组（17．1％,P〈0．05）。（3）bFGF组不同浓度的bFGF（1．0、5．0、10．0、20．0．40．0、80．0及160．0ng／ml）培养下,HAEC细胞增殖率分别为18．0％、35．7％、43．0％、52．7％、67．4％、43．6％及30．5％。bFGF组5～80ng／ml的HAEC增殖效应均明显大于对照组（P〈0．05）。其中40ng／ml时的细胞增殖率最高（P〈0．05）。（4）TGF-β1组不同浓度的TGF-β1（0．1、0．2、0．4、0．8、1．6、3．2、6．4及12．8ng／ml）培养下,HAEC细胞增殖率分别为17．1％、15．1％、9．3％、6．2％、4．8％、3．6％、2．0％、1．2％。TGF-β1组0．8～12．8ng／ml的HAEC增殖效应明显小于对照组（P〈0．05）。结论通过纤维蛋白支架,HAEC能移行修复缺损部位。EGF、bFGF能促进HAEC的增殖,而TGF-β1则抑制HAEC的增殖。     

Generation of trophoblast-like cells from the amnion in vitro: A novel cellular model for trophoblast development

Despite the high incidence of trophoblast-related diseases, the molecular mechanism of inadequate early trophoblast development is still unclear due to the lack of an appropriate cellular model in vitro. In the present study, we reprogrammed the amniotic cells to be induced pluripotent stem cells (iPSCs) via a non-virus and non-integrated method and subsequently differentiated them into trophoblast-like cells by a modified BMP4 strategy in E6 medium. Compared with the previously studied trophoblast-like cells from ESCs, the iPSCs derived trophoblast-like cells behave similarly in terms of gene expression profiles and biofunctions. Also we confirmed the differentiating tendency from iPSCs to be syncytiotrophoblasts-like cells might be caused by inappropriate differentiating oxygen condition. Additionally, we preliminarily indicated in vitro “artificial” differentiation of iPSCs also undergoing a possible trophoblastic stem cell stage, as witnessed in vivo. In conclusion, we provided an in vitro cellular model to study early trophoblast development for specific individual, by using the feasible amnion.     

胶联羊膜移植治疗兔角膜深层损伤的研究

目的 探讨胶联羊膜治疗兔角膜深层损伤的效果,分析其促进角膜损伤愈合的机制.方法 新西兰大白兔45只,均以右眼作为实验眼,实验前采用裂隙灯显微镜检查排除角膜疾病,建立兔角膜深层损伤模型.采用单纯随机抽样法,将兔分为胶联羊膜组、普通双层羊膜组及损伤对照组,每组15只.前两组行嵌合式羊膜移植.术后观察记录羊膜溶解脱落时间,角膜荧光素染色,角膜混浊及新生血管等情况,并进行临床疗效评定.术后第7、14、28天,行苏木素-伊红染色组织病理学检查;原位末端标记法检测角膜基质凋亡细胞.两个实验组和对照组各时间点参数评分以及末端脱氧核苷酸转移酶介导的脱脲苷三磷酸缺口末端标记法(TUNEL)阳性细胞表达值分别进行正态性检验,方差齐性检验,采取的统计方法为两独立样本t检验.结果 胶联羊膜覆盖于角膜创面的时间(20.00±2.43)d,明显长于普通双层羊膜(13.15±2.68)d(t=8.470,P=0.000).术后第28天,胶联羊膜组角膜创面全部恢复正常厚度,角膜上皮荧光素染色均为阴性,角膜透明度高,新生血管较少.而普通双层羊膜和对照组尚有部分动物角膜荧光素小片浅层着色,角膜混浊明显,瘢痕致密,粗大新生血管长入角膜中央.胶联羊膜组和对照组术后第14、28天,角膜混浊和新生血管总评分比较,差异有统计学意义(术后第14天:胶联羊膜组2.62,对照组5.19,t=3.986,P=0.004;术后第28天:胶联羊膜组2.87,对照组4.78,t=3.608,P=0.007).整个观测期,胶联羊膜组损伤角膜无感染穿孔;而普通双层羊膜组和对照组分别有3例和2例,因角膜上皮迁延愈合导致感染穿孔.组织病理检查显示胶联羊膜组损伤角膜全部正常上皮化,基质胶原纤维排列整齐.在各观察时间点上,胶联羊膜组角膜基质凋亡细胞均明显少于对照组(术后第7天:t=8.153,P=0.000;术后第14天:t=9.693,P=0.000;术后第28天:t=14.050,P=0.000).而普通双层羊膜组与对照组比较,仅在术后第7天,差异有统计学意义(术后第7天:t=5.474,P=0.000).结论 胶联羊膜对兔角膜深层损伤具有良好的治疗效果,可显著促进角膜损伤愈合,抑制新生血管和瘢痕形成,抑制角膜基质细胞凋亡.

Abstract：

Objective To treat deep layer corneal damage using fibrin glue and amniotic memrbane transplant. Methods Forty-five rabbits were given deep lamellar keratectomies to cause deep layer corneal damage in their right eyes and were then randomly divided into 3 groups. The first group was given doublelayer amniotic membrane transplants where fibrin glue was used to connect the two layers of amniotic membrane. The second group was given double-layer amniotic membrane transplants where no fibrin glue was used. The third group was given no treatment. Clinical outcome was graded by corneal integrity, opacity and neovascularization, and detachment of amniotic membrane was recorded. The expression of apoptosis was monitored to assess the changes of morphology and histology on the 7th, 14th and 28th days after surgery.Results While the double-layer amniotic membrane without fibrin glue covered the cornea for (13. 15 ±2. 68 ) d, the double-layer amniotic membrane using fibrin glue covered the surface of the cornea for (20. 00 ± 2. 43 ) d ( t = 8. 470, P = 0. 000 ). The corneas in the first group recovered smoothly and transparently, maintained normal thickness and less neovascularization, whereas the corneas in the other two groups recovered irregularly, lost their transparency, became turbid and showed higher levels of neovascularization. There were statistically significant differences between the first and third groups for corneal opacity and neovasculariazation, where the 14th day after surgery, the first group scored 2. 62 and the third group scored 5. 19 (t =3. 986, P =0. 004), and the 28th day after surgery, the first group scored 2. 87 and the third group scored 4. 78 (t =3. 608, P=0. 007). Perforation did not appear in the first group,but the second group had 3 cases and the third group had 2 cases, all due to infection. The changes of morphology and histology showed that the damaged corneas were contained normal epithelial cells and regularly arranged fibrous cells on the 28th day after surgery. The apoptosis in corneas of the first group was less than that of the third group at all observed points (7th day: t =8. 153, P =0.000; 14th day: t =9. 693, P =0. 000; 28th day: t = 14. 050, P =0. 000), however, apoptosis in corneas in the second group was only different from that in the third group on the 7th day after surgery while other observed points showed no difference (7th day: t =5.474, P=0. 000). Conclusion Using bio-engineered fibrin glue in amniotic membrane transplants can repair deep layer corneal damage, reduce neovascularization, scarring and corneal apoptosis.     

建立血管壁模型的一种新方法及其应用

为探讨建立血管壁模型的新方法,采用胰蛋白酶和胶原酶处理人胎儿羊膜,将人血管内皮细胞及平滑肌细胞分别培养在羊膜的两侧面,用联胺诱发脂质过氧化,观察单核细胞迁移的情况。结果显示,内皮细胞及平滑肌细胞可分别培养在经过处理的羊膜的两面,以构建成类似于机体的血管壁模型。     

用微核试验评价生物羊膜遗传毒性的研究

目的：用微核试验评价生物羊膜引起遗传毒性的可能性。方法参照《GB／T16886．3-1997／ISO10993-3：1992》的方法及指导原则进行试验。结果：在研究过程中所有动物临床表现和体重变化都是正常的，生物羊膜浸提液与阴性对照嗜多染红细胞（PCEs）／红细胞（RBc）总数无显著性差异（P〉0．05），没有出现遗传毒性的迹象。结论：生物羊膜用微核试验评价不会引起遗传毒性。     

Amnion as a prosthetic material in congenital defects

In pediatric surgery, amniotic membranes taken from autologous placenta are occasionally used as an implant in cases of large ventral abdominal clefts. The questions arise, which part of this organ should be used and how to use it in the recipient organism. Amniotic membranes consist anatomically of amnion and chorion, which are of fetal origin, and maternal decidua. In our experimental studies, we used the fetal parts of the amniotic membrane as an implant in a standardized rat model and investigated the utilization and possible foreign-body reaction (FBR) induced. Fifteen, 30, and 90 days after implantation the macroscopic appearance, light microscopy, and immunohistology of the specimens were examined. Adhesions to parenchymal organs and omentum were present irrespective of the side facing the abdominal cavity. Amnion induced a rapid FBR that diminished with time. Chorion and parts of the amnion were resorbed within the examined period after infiltration with recipient cells and neovascularization. Our studies have shown that for best results, only amnion in its anatomical definition and parts of the chorion should be prefered as an implant.