Objective To investigate a new composite matrix (BMSCs seeded on the denuded human amniotic membrane, BMSCs-DHAM) bridging the both stumps of spinal cord injury in rats to promote axon regeneration and improve motor function of hind l imbs. Methods The human amniotic membrane (HAM) was voluntarily donated by the healthy pregnant women after a caesarean section. The cells on the HAM were completely removed with a tryptic and mechanical approach to prepare DHAM. The BMSCs were separated and cultured from 4-week-old female rats (n=4), then the forth passage of BMSCs were labeled by PKH26 and seeded on DHAM (BMSCs-DHAM). The growing state of BMSCs was observed under themicroscopy. Moreover, 40 female rats (8-week-old, weighting 200-220 g) were made spinal cord injury models by transecting at T9 level, and were randomly divided into 4 groups (each group, n=10). The both stumps were respectively wrapped by BMSCs- DHAM or simple DHAM in groups A and C, and the same dose of BMSCs or physiological sal ine were also respectively injected the central lesion in groups B and D. At 12 weeks after surgery, the functional recovery of the hindl imbs was evaluated by the BBB locomotor rating score, and other indexes were tested including cortical motion evoked potential (MEP), anterograde biopinylated dextan amine (BDA) tracing, and immunofluorescence of neurofilament protein 200 (NF-200). Results HE staining proved that the DHAM was devoid of cellular components by this way, and BMSCs grew well on the substrate under the microscopy. At 12 weeks after operation, the BBB score (12.50 ± 1.26) in group A was significantly higher than those of other groups (P lt; 0.05), and the recovery in latency (3.52 ± 2.45) ms and ampl itude (480.68 ± 18.41) μV of MEP was also obviously improved in group A (P lt; 0.05) when compared with other groups. In addition, anterograde BDA tracing revealed that the rate of the positive BDA axons 54.12% ± 3.30% under the lesion level in group A was higher than those of other groups (P lt; 0.05), and lots of the regeneration axons (positive NF-200) were found to grow into the spinal cord under the composite matrix in group A. Conclusion The BMSCs-DHAM composite matrix can improve hindl imb motor function to some extent after spinal cord injury. It will be widely appl ied as the matrix material in the future.
Objective To investigate the effects of human acellularamnion membrane on SD rat tendon adhesion and to obtain the experimental data for clinical application in preventing postoperative tendon adhesion. Methods The tendons of 28 adult SD rats hindlimb were cut and sutured. The tendons of left hindlimb were encapsulated by human accellular amnion membraneas the experimental group and the ones of the other side were not encapsulatedas control group. The rats were killed 1, 2, 4, 6, 8 and 12 weeks after operation. The results were evaluated grossly and histologically. Results There were no differences in healing of injury tendon and inflammatory response between the two groups. The anatomical and histological results showed the experimental group had less adhesion than the control group(Plt;0.05). Conclusion Human acellular amnion membrane can prevent adhesion of tendonwithout affecting tendon healing and is an optimal biological material to prevent tendon adhesion.
截止至2002年5月,现有早产治疗的临床证据如下: (1) 高危早产:在一些国家实施的RCT发现,在降低早产危险方面,加强产前保健与普通产前保健没有明显差异.包括5个RCT的1个系统评价发现,对有宫颈改变的妇女行宫颈环扎术有不同的结果,没有明确的结论.1个大样本的RCT发现,孕9~29周宫颈功能可能不全的妇女进行预防性宫颈环扎手术与不环扎相比,能明显降低早产(<33孕周),但也会明显增加产褥感染的危险.另外4篇较小样本的RCT发现,孕10~30周、具各种早产高危因素的妇女,进行预防性宫颈环扎手术与不环扎相比,并不能降低早产(<34孕周).1篇系统评价的2个RCT报告,对有宫颈改变的妇女进行环扎术有不同的结果,其中1个RCT发现其并不能明显降低早产(<34孕周),而另外1个较小样本的RCT却发现宫颈环扎手术加卧床休息与单纯卧床休息比较,能明显降低34周前的早产.没有1个RCT证实行环扎术加卧床休息与单纯卧床休息相比,能降低围生儿死亡率. (2) 胎膜早破:1个系统评价发现,对胎膜早破的妇女,抗生素较安慰剂能明显延长孕周、降低新生儿发病率的危险,如新生儿感染、出生后氧疗、脑部超声异常等.阿莫西林加克拉维酸治疗与新生儿坏死性小肠结肠炎的发生率明显增加有关.一个基于1个RCT的系统评价发现,没有充足的证据证实羊膜腔灌注与不灌注比较能改善胎膜早破后的新生儿结局. (3) 先兆早产的治疗:①β-肾上腺素兴奋剂:1个系统评价发现,β-肾上腺素兴奋剂与安慰剂或不治疗相比,并不能明显降低围生儿死亡率、呼吸窘迫综合征及低体重儿(<2 500 g)发生率,且与与安慰剂或不治疗相比,β-肾上腺素兴奋剂增加孕母副反应,如胸痛、心悸、呼吸困难、震颤、恶心、呕吐、头痛、高血糖、低钾血症.②钙离子通道拮抗剂: 没有关于钙离子通道拮抗剂与安慰剂比较的系统评价或RCT.1个系统评价发现,钙离子通道抑制剂与其它保胎药(主要是β-肾上腺受体兴奋剂)比较,能显著降低48 h内的早产分娩,减少因孕母副反应退出治疗和新生儿发病率.③硫酸镁:1个系统评价发现,硫酸镁与安慰剂比较,并不能明显降低孕36周前的早产率、围生儿死亡率、呼吸窘迫综合征的发生率.另一个系统评价发现,硫酸镁和其他宫缩抑制剂(β-肾上腺素兴奋剂、钙离子通道拮抗剂、前列腺素合成抑制剂、硝化甘油、酒精和葡萄糖注射剂)比较,并不能明显降低48 h内早产率(尽管结果没有差异).④垂体受体拮抗剂(阿托西班):1个系统评价纳入 2个RCT,对阿托西班和安慰剂治疗早产进行比较有不同的结果.较大样本的RCT发现,阿托西班较安慰剂能延长孕周,但阿托西班增加了孕28周以下的胎儿死亡率.另一个RCT发现,阿托西班增加了48 h内的早产.⑤前列腺素抑制剂(消炎痛):1个系统评价发现,消炎痛与安慰剂比较,能明显降低孕37周前的48 h和7天的早产率的证据有限.然而,同时发现消炎痛与安慰剂或不治疗相比,并不能明显降低围生儿死亡率、新生儿呼吸窘迫综合征、肺支气管发育不良、坏死性小肠结肠炎、新生儿败血症或低体重儿.但这个系统评价样本太小,尚不能发现有临床意义的差异. (4) 择期或非择期剖宫产对早产妇女治疗效果:1个系统评价结果发现,择期剖宫产较非择期剖宫产会增加孕母的发病率,却不能降低新生儿的发病率和死亡率.但尚不能证明此效果是否对新生儿有临床意义. (5) 改善早产妊娠结局的干预措施:①对早产者采用皮质类固醇:1个系统评价认为,对可能发生早产的妇女使用皮质激素较安慰剂或不处理能明显降低早产儿出生后呼吸窘迫综合征、新生儿死亡率和颅内出血的发生.②促甲状腺激素释放激素在早产中的运用:1个系统评价发现,在早产的高危妇女中,促甲状腺激素释放激素和类固醇激素联合应用与单用皮质类固醇激素比较,对新生儿结局的影响无明显差异,但会明显增加孕母和胎儿的不良反应.③抗生素:1个系统评价发现,抗生素与安慰剂比较,不能延长孕周、降低新生儿死亡率,但可降低孕母感染率.
Objective To study the differentiation of the human osteoblasts during the construction of the tissue engineered periosteum with the human acellular amniotic membrane(HAAM).Methods To construct the tissue engineered periosteum (n=60) with HAAM, the human fetal osteoblasts were used. The fetal osteoblasts were cultured for 2, 4, 6, 8, and10 days, and then their total RNA was extracted, which were reversely transcripted to cDNA. The realtime PCR analysis was used to reveal Cbfal and Osterix, and the cycle threshold (Ct) was also measured. The simplycultured osteoblasts were used as the control group (n=20).Results The expression of Cbfa1 was higher in the experimental group on the 2nd day when compared with that on the 4th, 6th, and 8th day(P<0.05). The same result existed on the 10th day when compared with that on the 4th and 8th day. The expression of Osterix increased and was highest on the 8th day when compared with the other results(P<0.05). Both of the 2 gene expressions were decreased in the control group when compared with those in the experimental group, but with no significant difference(P>0.05). Conclusion Cbfa1 and Osterix can be normally expressed by the osteoblasts after their integration with HAAM. As a scaffold, HAAM can be used to keep the osteoblast phenotype and differentiation with an osteoconductive ability. Such a cell-scaffold complex may provide a basis for the osteogenesis.
Objective To evaluate the clinical efficiency of fresh amniotic membrane transplantation in treatment of stenosis of conjunctival sac. MethodsThirty cases (30 eyes) of stenosis of conjunctival sac were treated with fresh amniotic membrane transplantation. Amniotic membrane was obtained under sterile conditions after elective cesarean delivery. The woman’s serum was negative for HBsAg, syphilis, and human immunodeficiency virus. The placenta was first washedfree of blood clots with sterile saline. Under sterile conditions, the inner amniotic membrane was separated from the chorion by blunt dissection, and was cleaned of blood with the sterile saline again. The membrane was then flattened onto a surgidrape adhesive paper with the epithelium surface up. The paper with the adherent amniotic membrane was then cut into 5 cm×8 cm pieces, and then rinsed in solution containing 4×106 U/L of gentamycin and stored at 4℃. It could bestored for 12 hours after preparation. The-adhesiotomy was performed firstly. The separation between the conjunctiva and scar tissue should be complete and wide enough to reach to the orbital margin. The adhesiectomy was taken secondly. The scar tissues were removed completely. The fresh amniotic membrane was flattened onto the conjunctival defect with epithelium side up. The fresh amniotic membranewas 10 mm more than the conjunctival defect by trimming off the excess portion.This fashioned membrane was then secured to surrounding conjunctival edge with continuous 7-0 nylon sutures. The necessary mattress suture of inferior conjunctival fornix via skin next to the inferior orbital margin was performed simultaneously. The retrobular implantation of the an artificial globe made of hydroxyapatite was performed on some patients with sunken eye. Correction of traumatic ptosis was performed on a few patients.Results The operation ofreconstruction of partial conjunctival sac for 30 cases was successful. All amniotic membrane grafts were alive. The cosmetic result was complete favorable. The infection and contracture of the graft, immunologic rejection and amniotic lysis were not observedin all cases during the follow-up period of 13-18 months.Conclusion Fresh amniotic membrane transplantation can be used in reconstruction of the partial conjunctival sac effectively and can be popularized in thelocal hospital in China because the amniotic membrane can be obtained easily.
Objective Inducing human amniotic membrane mesenchymal stem cells (hAMSCs) to Schwann cells-like cells (SCs-like cells) in vitro, and to evaluate the efficacy of transplantation of hAMSCs and SCs-like cells on nerves regeneration of the rat flaps. Methods hAMSCs were isolated from placenta via two-step digestion and cultured by using trypsin and collagenase, then identified them by flow cytometry assay and immunofluorescence staining. The 3rd generation of hAMSCs cultured for 6 days were induced to SCs-like cells in vitro; at 19 days after induction, the levels of S-100, p75, and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence staining, Western blot, and real-time fluorescence quantitative PCR (qPCR). The levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were measured by ELISA in the supernatant of the 3rd generation of hAMSCs cultured for 6 days and the hAMSCs induced within 19 days. In addition, 75 female Sprague Dawley rats were taken to establish the rat denervated perforator flap model of the abdominal wall, and were divided into 3 groups (n=25). The 3rd generation of hAMSCs (1×106 cells) in the proliferation period of culturing for 6 days, the SCs-like cells (1×106 cells), and equal volume PBS were injected subcutaneously in the skin flap of the rat in groups A, B, and C, respectively. At 2, 5, 7, 9, and 14 days after transplantation, 5 rats in each group were killed to harvest the flap frozen sections and observe the positive expression of neurofilament heavy polypeptide antibody (NF-01) by immunofluorescence staining. Results The cells were identified as hAMSCs by flow cytometry assay and immunofluorescence staining. The results of immunofluorescence staining, Western blot, qPCR showed that the percentage of positive cells, protein expression, and gene relative expression of S-100, p75, and GFAP in SCs-like cells group were significantly higher than those in hAMSCs group (P<0.05). The results of ELISA demonstrated that the expression of BDNF and NGF was significantly decreased after added induced liquid 1, and the level of BDNF and NGF increased gradually with the induction of liquids 2 and 3, and the concentration of BDNF and NGF was significantly higher than that of hAMSCs group (P<0.05). Immunofluorescence staining showed that the number of regenerated nerve fibers in group B was higher than that in groups A and C after 5-14 days of transplantation. Conclusion The hAMSCs can be induced into SCs-like cells with the proper chemical factor regulation in vitro, and a large number of promoting nerve growth factor were released during the process of differentiation, and nerve regeneration in flaps being transplanted the SCs-like cells was better than that in flaps being transplanted the hAMSCs, which through a large number of BDNF and NGF were released.
Objective The human amniotic epithel ial cells (hAECs) are a recently identified new type of stem cells.It has previously been shown that hAECs express hepatocyte-related gene and possess intracellular features and functional properties of hepatocytes. The hAECs may be a candidate seed cell for l iver regeneration. To research the survival and migrationin vivo of hAECs via adeno-associated virus-mediated the green fluorescent protein gene (AAV-GFP) transfection, and toexplore the expression of hepatocyte-l ike function. Methods Thirty nude mice (aging 6-8 weeks, half males and females, and weighing 20-22 g) were randomly divided into 3 groups (groups A, B, and C, n=10). The mice of groups A and C were made the 2/3 partial hepatectomy model, and the mice of group B underwent open abdominal operation without hepatectomy. The hAECs transfected by AAV-GFP were transplanted into the inferior end of the spleen in groups A and B with a cell density of 5 × 106/mL and a volume of 0.2 mL; the same volume of normal sal ine was injected in group C. At 4 hours, the nude mice were sacrificed and the samples of l iver, spleen, heart, lung, brain, and kidney were harvested and the general observation, histological observation, and immunofluorescence detection were performed for the hAECs survival, migration, and the functional properties of hepatocytes. Results No tumor tissue was found in l iver and spleen of 3 groups, and HE staining showed no tumor cells. There were a lot of roundl ike and deeply-stained cells with less cytoplasm and large nucleus in the spleen and the l iver of group A; no abnormal cells were found in l iver and spleen of groups B and C and in kidney, heart, bung, and brain of groups A, B, and C. The GFP+ cells were detected in the spleen and l iver of group A with expressing human albumin, but no GFP+ cells was found in l iver and spleen of groups B and C and in heart, kidney, lung, and brain of groups A, B, and C. Conclusion AAV-GFP infected hAECs transplanted into SCID nude mice with hepatectomy can keep the hepatocyte-l ike function. It will be beneficial to further identify their biological characteristics.
目的 观察两种手术方式治疗翼状胬肉的临床效果。 方法 对2007年1月-2012年6月一般情况较好的73例单纯原发性双眼翼状胬肉患者进行了回顾性研究,比较同一患者双眼分别行翼状胬肉切除+生物羊膜移植(左眼)和翼状胬肉切除+自体角膜缘干细胞移植(右眼)手术后痊愈率、复发率及并发症。 结果 行翼状胬肉切除+自体角膜缘干细胞移植(右眼)的痊愈率达93.15%,复发率1.37%,并发症率5.48%,其痊愈率更高而复发率及并发症均更低。 结论 翼状胬肉切除+自体角膜缘干细胞移植用于治疗翼状胬肉比翼状胬肉切除+人工羊膜移植的疗效更好,值得临床推广。
Objective To observe the differentiation effect of rabbit amnion-derived stem cells (ADSC) induced into neural cells.Methods ADSC of New Zealand female rabbits were isolated and cultured. Its mRNA level of Fibronectin, Nestin and Vimentin were detected by real-time quantitative polymerase chain reaction. The selfreplication ability of ADSC was confirmed by monoclonal formation experiments. These ADSC were further induced into neural cells in vitro. Five days after induced differentiation, the expression of -tubulin and glial fibrillary acidic protein (GFAP) were detected by immunofluorescent staining. Results ADSC were separated from amnion tissue gradually after 24 hours. There were polygonal cells gathered around the amnion tissue at 72 hours, and were distributed compactly around the amnion at 120 hours. The morphology of cleavage daughter cells was basically the same as parent cells. ADSC has the ability of self-replication. The Nestin, Vimentin, Fibronectin mRNA expressions in ADSC were 15.79, 1.91, 7.65 times those in spleen cells. The differences were statistically significant(Z=-5.243, -3.972, -2.524; P<0.05). The beta;-tubulin expression was found in cytoplasm of most cells. The GFAP expression was found in cytoplasm in some cells. Conclusions ADSC has self-replication ability. It can be induced into neurons and neuroglial cells under the right conditions.