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.
目的:觀察新鮮羊膜移植聯合絲裂霉素C治療復發性翼狀胬肉的臨床療效。方法:對32例(38眼)復發性翼狀胬肉行翼狀胬肉切除聯合新鮮羊膜移植加絲裂霉素C治療,觀察術后角膜上皮愈合、胬肉復發情況。結果:術后隨訪3～24個月,有2眼復發,復發率為5.26%。結論:新鮮羊膜移植聯合絲裂霉素Ｃ治療復發性翼狀胬肉降低了復發率,無嚴重手術并發癥,是一種安全有效的手術方法。
Objective To review the latest development of amniotic membrane andits application. Methods Related literatures on the development of amniotic membrane and its application were extensively reviewed and summarized. Results There were amniotic epithelial cells and many growth factors in the outer layer of amniotic membrane and there were many kinds of collagen in the basement. The special structure promoted the growth of many kinds of cells. It was widely used in ophthalmology. Conclusion As it is easily available, compatible, cheap in price, low in antigenicity, and able to promote the growth of many kinds of cells, with few ethical problems involved, amniotic membrane will be more and more widely applied.
【摘要】 目的 探討延期妊娠的結局及防治。 方法 回顧性分析2008年6月-2009年6月收治的1 157例延期妊娠臨床資料，根據妊娠時段分A、B、C三組，A組449例，妊娠40+1~40+3周；B組358例，妊娠40+4~40+6周；C組350例，妊娠41~41+6周。比較各組羊水糞染發生率，剖宮產率，新生兒轉歸情況。 結果 隨妊娠時段的延長，羊水糞染發生率，剖宮產率具有統計學意義的變化（Plt;0.05）。新生兒評分低，轉專科治療的新生兒增多。 結論 延期妊娠為高危妊娠，應加強監護及檢測手段，適時終止妊娠。【Abstract】 Objective To explore the outcome of prolonged pregnancy and treatment. Methods Clinical data of 1 157 cases of prolonged pregnancy were retrospectively analyzed during June 2008 to June 2009.They were divided into three groups according to the time of pregnancy.Group A: 449 cases, pregnant age 40+1 - 40+3 week; Group B:358 cases, pregnant age 40+4 - 40+6 weeks; Group C:350 cases, pregnant age 41 - 41+6 weeks. The incidence of amniotic fluid turbidity, the rate of cesarean section and the neonatal prognosis were compared among three groups. Results With the extension of time of pregnancy, the incidence of amniotic fluid turbidity and the rate of cesarean section were statistically different among three groups (Plt;0.05), neonatal score was low, and the number of cases who needed specialist treatment increased. Conclusion Prolonged pregnancy is a high-risk pregnancy.The monitoring and detection means for prolonged pregnancy should be strengthened.Termination of pregnancy should be considered if necessary.
ObjectiveTo evaluate the safety and efficacy of amniotic membrane patching in the treatment of recurrent macular hole associated with retinal detachment of high myopia (MHRD). MethodsA prospective study. From March 2018 to January 2020, 11 patients (11 eyes) of recurrent macular hole associated with MHRD at the First Affiliated Hospital of Zhengzhou University were enrolled. Among them, there were 3 males (3 eyes), and 8 females (8 eyes). The average age was 63.64±5.82. The axis length (AL) was 29.10±0.59 mm, and the logarithm of the minimum angle of resolution best corrected visual acuity (logMAR BCVA) was 2.23±0.57. Patients previously received pars plana vitrectomy (PPV) combined with internal limiting membrane stripping surgery, which was more than 1 time. All eyes underwent standard pars plana three-channel 23G PPV combined with amniotic membrane covering and silicone oil filling. The silicone oil was removed 6 months after surgery. Follow-up time was up to 3 months after silicone oil removal surgery. 1, 3, and 6 months after the operation, the same equipment and methods were used to conduct relevant examinations before the operation to observe the closure of the macular hole, retinal reattachment and changes in logMAR BCVA. The logMAR BCVA before and after surgery was compared by paired t test. ResultsAt 1, 3, and 6 months after the operation, the retinas of all eyes were anatomically repositioned, the macular holes were well closed, and the amniotic membrane was attached to the retina. At 3 months after the silicone oil removal operation, there was no recurrence of macular hole in all eyes; logMAR BCVA was 1.35±0.32. No serious complications occurred during and after surgery in all eyes. ConclusionAmniotic membrane patching is a safe and effective method for recurrent macular hole associated with MHRD.
Objective To study the vascularization of the compositeof bio-derived bone and marrow stromal stem cells(MSCs) in repairing goat tibial shaft defect.Methods Bio-derived bone was processed as scaffold material. MSCs were harvested and cultured in vitro. The multiplied and induced cells were seeded onto the scaffold to construct tissue engineered bone. A 20 mm segmental bone defect inlength was made in the middle of the tibia shaft in 20 mature goats and fixed with plate. The right tibia defect was repaired by tissue engineered bone (experimental side), and the left one was repaired by scaffold material (control side).The vascularization and osteogenesis of the implants were evaluated by transparent thick slide, image analysis of the vessels, and histology with Chinese ink perfusion 2, 4, 6, and 8 weeks after operation.Results More new vessels were found in control side than in experimental side 2 and 4 weeks after implantation (Plt;0.05). After 8 weeks, there was no significant difference in number of vessels between two sides(Pgt;0.05), and the implants were vascularized completely. New bone tissue was formed gradually as the time and the scaffold material degraded quickly after 6 and 8 weeks in the experimental side. However, no new bone tissue was formed andthe scaffold degraded slowly in control side 8 weeks after operation.Conclusion Bio-derived bone has good quality of vascularization. The ability of tissue-engineered bone to repair bone defect is better than that of bio-derived bone alone.
Objective To investigate whether the Runx2 gene can induce the differentiation of human amniotic mesenchymal stem cells (hAMSCs) to ligament fibroblasts in vitro and promote the tendon-bone healing in rabbits. Methods hAMSCs were isolated from the placentas voluntarily donated from healthy parturients and passaged, and then identified by flow cytometric identification. Adenoviral vectors carrying Runx2 gene (Ad-Runx2) and empty vector adenovirus (Ad-NC) were constructed and viral titer assay; then, the 3rd generation hAMSCs were transfected with Ad-Runx2 (Ad-Runx2 group) or Ad-NC (Ad-NC group). The real-time fluorescence quantitative PCR and Western blot were used to detect Runx2 gene and protein expression to verify the effectiveness of Ad-Runx2 transfection of hAMSCs; and at 3 and 7 days after transfection, real-time fluorescence quantitative PCR was further used to detect the expressions of ligament fibroblast-related genes [vascular endothelial growth factor (VEGF), collagen type Ⅰ, Fibronectin, and Tenascin-C]. The hAMSCs were used as a blank control group. The hAMSCs, hAMSCs transfected with Ad-NC, and hAMSCs were mixed with Matrigel according to the ratio of 1 : 1 and 1 : 2 to construct the cell-scaffold compound. Cell proliferation was detected by cell counting kit 8 (CCK-8) assay, and the corresponding cell-scaffold compound with better proliferation were taken for subsequent animal experiments. Twelve New Zealand white rabbits were randomly divided into 4 groups of sham operation group (Sham group), anterior cruciate ligament reconstruction group (ACLR group), anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-NC-scaffold compound group (Ad-NC group), and anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-Runx2-scaffold compound group (Ad-Runx2 group), with 3 rabbits in each group. After preparing the ACL reconstruction model, the Ad-NC group and the Ad-Runx2 group injected the optimal hAMSCs-Matrigel compunds into the bone channel correspondingly. The samples were taken for gross, histological (HE staining and sirius red staining), and immunofluorescence staining observation at 1 month after operation to evaluate the inflammatory cell infiltration as well as collagen and Tenascin-C content in the ligament tissues. ResultsFlow cytometric identification of the isolated cells conformed to the phenotypic characteristics of MSCs. The Runx2 gene was successfully transfected into hAMSCs. Compared with the Ad-NC group, the relative expressions of VEGF and collagen type Ⅰ genes in the Ad-Runx2 group significantly increased at 3 and 7 days after transfection (P<0.05), Fibronectin significantly increased at 3 days (P<0.05), and Tenascin-C significantly increased at 3 days and decreased at 7 days (P<0.05). CCK-8 detection showed that there was no significant difference (P>0.05) in the cell proliferation between groups and between different time points after mixed culture of two ratios. So the cell-scaffold compound constructed in the ratio of 1∶1 was selected for subsequent experiments. Animal experiments showed that at 1 month after operation, the continuity of the grafted tendon was complete in all groups; HE staining showed that the tissue repair in the Ad-Runx2 group was better and there were fewer inflammatory cells when compared with the ACLR group and the Ad-NC group; sirius red staining and immunofluorescence staining showed that the Ad-Runx2 group had more collagen typeⅠ and Ⅲ fibers, tending to form a normal ACL structure. However, the fluorescence intensity of Tenascin-C protein was weakening when compared to the ACLR and Ad-NC groups. Conclusion Runx2 gene transfection of hAMSCs induces directed differentiation to ligament fibroblasts and promotes tendon-bone healing in reconstructed anterior cruciate ligament in rabbits.
Objective To study the preparation method of acellular vascular matrix and to evaluate its biocompatibil ity and safety so as to afford an ideal scaffold for tissue engineered blood vessel. Methods Fresh caprine carotids (length, 50 mm) were harvested and treated with repeated frozen (—80 )/thawing (37℃), cold isostatic pressing (506 MPa, 4 ), and 0.125% sodium dodecyl sulfate separately for preparation of acellular vascular matrix. Fluorescence staining and DNA remain test were used to assess the cell extracting results. Biological characteristics were compared with the raw caprine carotids using HE staining, Masson staining, scanning electron microscope (SEM), and mechanical test. Biocompatibil ity wasdetected using cell adhesion test, MTT assay, and subcutaneously embedding test. Ten SD rats were divided into 2 groups (n=5). In experimental group, acellular vascular matrix preserved by the combination of repeated frozen/thawing, ultrahigh pressure treatment and chemical detergent was subcutaneously embedded; and in control group, acellular vascular matrix preserved only by repeated frozen/thawing and ultrahigh pressure treatment was subcutaneously embedded. Results HE staining and Masson staining revealed that no nucleus was detected in the acellular vascular matrix. SEM demonstrated that a lot of collagen fibers were preserved which were beneficial for cell adhesion. Fluorescence staining and DNA remain test showed that the cells were removed completely. There was no significant difference in stress and strain under the maximum load between before and after treatment. Mechanical test revealed that the acellular vascular matrix reserved mechanical properties of the raw caprine carotids. Cell adhesion test and MTT assay confirmed that cytotoxicity was grade 0-1, and the acellular vascular matrix had good compatibil ity to endothel ial cells. After subcutaneously embedding for 8 weeks, negl igible lymphocyte infiltration was observed in experimental group but obvious lymphocyte infiltration in control group. Conclusion The acellular vascular matrix, which is well-preserved by the combination of repeated frozen/thawing, ultrahigh pressure treatment, and chemical detergent, is an ideal scaffold for tissue engineered blood vessel.
Objective To study the method to prepare the animal model of goat cleft palate by injection of anabasine and the effect of the malformation on the development of the facial mid-part. Methods A total of 40 female boer hybrid goats were selected, aging 8-12 months and weighing 35-55 kg. The mating day was 0 day, and at 30 days the goats assured pregnant byB type ultrasonic test were divided into 4 groups (n=10) according to intramuscular injection of 10 (experimental group 1), 15 (experimental group 2), 20 (experimental group 3) mg/ d, and no injection (control group), respectively, from the 31st to 42nd day. At pregnant 120 days and 1 month after birth, 5 fetal goats of each group were used for three dimensional reconstruction ofskull with CT scan. The maxillary bone width named as PPMM and the maxillary bone length named as APMM were measured then the hard palate general observation was performed and dry skull of goats was harvested to observe the development of maxillary. Results After injection, all pregnant lambs aborted in experimental group 3; 2 pregnant lambs aborted and 8lambs maintained pregnancy in experimental group 2. At 120 days of pregnant, no cleft palate was observed in 5 fetal lambs of experimental group 1 and control group, respectively; cleft palate and maxillary dysplasia occurred in 3 fetal lambs of experimental group 2. Among 11 newborn lambs of experimental group 1 and 8 newborn lambs of control group, no cleft palate was observed;among 7 newborn lambs of experimental group 2, cleft palate occurred in 5 with obvious maxillary dysplasia and eating difficultly. General observation of hard palate and dry skull showed obvious hypoplasia of maxillary in experimental group 2. There were significant differences in PPMM and APMM between the experimental group 2 and the control group at pregnant 120 days and 1 month after birth (P lt; 0.05). Five lambs with cleft palates of experimental group 2 survived for 1-2 months. Conclusion The animal models of goat cleft palate can established by intramuscular injection of anabasine at a dose of 15 mg/d from the 31st to 42nd day of pregnant. The facial character of the induced cleft palate goat is similar to that of human cleft palate.