目的 觀察兩種手術方式治療翼狀胬肉的臨床效果。 方法 對2007年1月－2012年6月一般情況較好的73例單純原發性雙眼翼狀胬肉患者進行了回顧性研究，比較同一患者雙眼分別行翼狀胬肉切除+生物羊膜移植（左眼）和翼狀胬肉切除+自體角膜緣干細胞移植（右眼）手術后痊愈率、復發率及并發癥。 結果 行翼狀胬肉切除+自體角膜緣干細胞移植（右眼）的痊愈率達93.15%，復發率1.37%，并發癥率5.48%，其痊愈率更高而復發率及并發癥均更低。 結論 翼狀胬肉切除+自體角膜緣干細胞移植用于治療翼狀胬肉比翼狀胬肉切除+人工羊膜移植的療效更好，值得臨床推廣。
Objective To explore an effective method to culture and purify porcine keratinocytes, to observe the morphological characteristics of porcine keratinocytes growing on acellular amnion and to offer the experimental basis for that the amnion is used for tissue engineering. Methods The primary porcine keratinocytes were cultivated with DKSFM(Defined keratinocyteSFM) containing 10% fetal bovine serum (FBS). The second passage porcine keratinocytes were cultivated with the medium of DKSFM containing different concentrations of FBS. Because of the speciality that keratinocytes stick to flask fast, we purified the keratinocytes by 0.02% EDTA and 005% trypsin step by step. The second passage keratinocytes were seeded on amnion, the keratinocytes/amnion composites were observed by dye directly, histopathology and immunohistochemical staining. Results The proliferation of the primry porcine keratinocytes cultured with the medium ofDKSFM containing 10% FBS was fast and the morphological characteristics were good. The cultivated porcine keratinocytes expanded to 60%70% of the total area of the bottle of the flask after 5 days. The proliferation of the second passage porcine keratinocytes cultivated with the medium that DKSFM containing 5% FBS was faster than the second porcine keratinocytes cultured with the medium of DKSFMcontaining 10% FBS, or DKSFM without FBS. The proliferation of the second passage porcine keratinocytes cultivated with DKSFM without FBS was the slowest one among the 3 medium. The porcine keratinocytes that were purified by 0.02% EDTA and 005% trypsin step by step were got with high pure. After the keratinocytes were cultivated on the surface of amnion 12 days, the keratinocytes form a single layer on the surface of amnion and the cells were polygong and arranged like slabstone. After 14 and 16 days,the cells contacted more closely. But at 16 days after the cells were seeded, some of the cells got aging. Conclusion To culture primary porcine keratinocytes with the medium that DKSFMcontaining 10% FBS and to cultivate the second passage with the medium containing 5% FBS, the proliferation of porcine keratinocytes are faster. The method that purify the porcine keratinocytes is effective. Acellular amnion offers excellent bioscafold to support keratinocytes to adhere and grow. After the porcine keratinocytes are cultivated on the surface of the acellular amnion 12 days, the morphologic characteristics are better than that of other groups.
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.
Objective To observe clinical effects of burn wounds treatment with bovine amnion and to screen the best method of preparing and storing of bovine amnion. Methods From January 2004 to January 2005,We selected randomly 58 patients with superficial Ⅱ° wound, deepⅡ° wound, autografting area for removal of eschars and tangential excision, fetching skin area or residual burn wound . Using auto-control, every burn wound was divided into 3 parts and was treated with 3 dressings: bovine amnion dealt with by 0.1% chlorhexidine（group A）, bovine amnion dealt with by 0.4% glutaraldehyde（group B） and vaseline gauze dressing（group C as control）. The clinical effects were compared between different groupsand the method of preparing and storing bovine amnion was evaluated. Results The dressing texture of group A was softer than that of group B, and its flexibility was fine. The pretreatment was not necessary for dressing in group A. When the dressing was used on burn wounds in groups A and B, painwas slight, but pain was obvious in group C; healing time in groups A and B was much less than that in group C, showing statistically significant difference(P＜0.01). There was no statistically significant difference in healing time between groups A and B (P＞0.05). The infection ratio of burn wound in deepⅡ° wound and residual burn wound of groups A and B is much lower than that of group C, showing statistically significant difference (P＜0.05); in theother burn wounds there was no significant difference (P＞0.05). There was no statistically significant difference between groups A and B (P＞0.05). Conclusion Bovine amnion could make benefit on burn wounds healing, reduce infection ratio of burn wounds, could be used on different kinds of burn wounds. The clinical effect between bovine amnion dealt with by glutaraldehyde and by chlorhexidine is similar. Whereas the latter is more easy to be popularized.
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.
Objective To investigate the clinical therapeutic effects of two types of vaginoplasty. Methods From January 1996 to March 2005, 63 patients wih the congenital absence of the vagina were treated by two types of vaginoplasty. Of the 63 patients, 37 underwent vaginoplasty using the amnion and 26 underwent an improved laparoscopic Vecchitti operation. The durations ofthe operation and hospitalization, as well as the blood loss were compared between the two types of vaginoplasty. The vaginal moulds were improved during the operations. Results According to the follow-up for 2 months to 4 years in the 35 patients. Compared with vaginoplasty using the amnion, vaginoplasty by an improved laparoscopic Vecchitti operation had advantages of significantly shorter surgical duration, shorter hospitalization, and less blood loss (Plt;0.05). After the operations, the artificial vagina of all the 63 patients could hold a speculum and the mucosa appeared so soft and smooth with normal lubrication. The married patients were satisfied with the intercourse. However, after vaginoplasty using the amnion, an infection of the amnion occurred in 3 patients, scar contracture in 2 patients, one of whom underwent scar incision 13 months after operation with a success; but the other refuse to accept another operation. But the improved laparoscopic Vecchitti operation achieved a success in the patients without any infectionor scar contracture, according to the 2 month-2.5 years follow-up. Conclusion The improved laparoscopic Vecchitti operation is a preferred procedure of constructing a vagina for the patients suffering from the congenital absence of the vagina.
Application research on human amniotic membrane has been carried out for nearly a hundred years and people found that there were more than dozens of kinds bioactive substances in the amniotic membrane. It has been proved that the amniotic membrane has a lot of functions, such as anti-inflammatory, anti-bacterial, anti-virus, anti-angiogenic and promoting cell apoptosis, and so on. As effective treatments, amniotic membrane has been used for adjunctive therapy of burns, trauma, ophthalmic damage, dermatopathya. Recent advances of amniotic membrane and amniotic membrane-derived cells research have led to enormous progress in skin tissue engineering, vascular tissue engineering, biological scaffold material, and biological sustained-release materials. Amniotic membrane and amniotic membrane derived cells have a significant advantage and unique charm in medical field. Therefore, they have higher research value and broad prospects in the applications.
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 To observe the effects of culture medium of amniotic cells on NO and NOS in retinal tissues of rabbits in vitro in order to provide a protective method for antioxidation in retina transplantation. Methods Thirty adult healthy rabbits (30 right eyes) were divided into 3 groups. Group I: fresh retinal tissue; group II: routine culture medium; group III: culture medium of amniotic cells. The retinal tissues in group II and III were cultured in the corresponding culture medium for 1 week. The content of NO and NOS in retinal tissues in the 3 groups were determined. Results Compared with group I, the content of NO and NOS of group II increased obviously (t=3.821, 3.854; P＜0.001). There was no statistical difference of content of NO and NOS between group I and III (t=1.657, 1.745; P＞0.05). Conclusion Culture medium of amniotic cells may remove free radicals and enhance the ability of antioxidation. (Chin J Ocul Fundus Dis，2004，20：366-368)
Objective To compare the reparative effects between the acellular small intestinal submucosa andthe acellular amnion as dressings for traumatic skin defects. Methods Three full-thickness skin defects, which wereclose to the vertebral column of the pig, were created on both sides of the dorsum. The skin defects were randomlydivided into three groups. In each group, the following different materials were used to cover the skin defects: theacellular amnion in Group A, the acellular small intestinal submucosa (SIS) in Group B, and the physiological saline aguze in Group C (the control group). The specimens from the skin defects were harvested for a histological evaluation and for determination of the hydroxyproline content at 10 (2 pigs), 20 (2 pigs), and 30 days (3 pigs). We observed the healing process of the wound and its healing rate, counted the inflammatory cells, vasecular endothelial cells, and proliferating cells, and determined the hydroxyproline content. Results The acellular amnion in Group A and acellular SIS in Group B adhered to the wound tightly, but they did not adhere to the dressing; when the dressing was changed, the wound did not bleed. The saline gauze in Group C adhred to the wound tightly, but when the dressing was changed, the wound bled until 22 days after operation. Under the microscope, the collagen in the tissue below the epithelium was arranged more regularly and there were fewer cells concerned with inflammation in Groups A and B than in Group C at 10, 20, and 30 days after operation. At 10, 20, and 30 days after operation, the wound healing rate was greater in Groups A and B than in Group C, The number of the inflammatory cells and the proliferating cells were greater in Groupo C than in Groups A and B. There was a statistically significant difference (P lt; 0.05),At 20 and 30 days after operatin, the content of hydroxyproline was greater in Group c than in Group A and B. There was a statistically significant difference (P lt; 0.05). However, there was no statistically significant difference between Group A and Group B in the wound healing rate, the numbers of the inflammatory cells, vascular endothelial cells and prokiferating cells, and the content of hydroxyproline(P gt; 0.050). There was no statistically significant difference among the three groups in the number of the vascular endothelial cells. Conclusion Compared with Group C........