截止至2002年5月,現有早產治療的臨床證據如下: (1) 高危早產:在一些國家實施的RCT發現,在降低早產危險方面,加強產前保健與普通產前保健沒有明顯差異.包括5個RCT的1個系統評價發現,對有宮頸改變的婦女行宮頸環扎術有不同的結果,沒有明確的結論.1個大樣本的RCT發現,孕9～29周宮頸功能可能不全的婦女進行預防性宮頸環扎手術與不環扎相比,能明顯降低早產(＜33孕周),但也會明顯增加產褥感染的危險.另外4篇較小樣本的RCT發現,孕10～30周、具各種早產高危因素的婦女,進行預防性宮頸環扎手術與不環扎相比,并不能降低早產(＜34孕周).1篇系統評價的2個RCT報告,對有宮頸改變的婦女進行環扎術有不同的結果,其中1個RCT發現其并不能明顯降低早產(＜34孕周),而另外1個較小樣本的RCT卻發現宮頸環扎手術加臥床休息與單純臥床休息比較,能明顯降低34周前的早產.沒有1個RCT證實行環扎術加臥床休息與單純臥床休息相比,能降低圍生兒死亡率. (2) 胎膜早破:1個系統評價發現,對胎膜早破的婦女,抗生素較安慰劑能明顯延長孕周、降低新生兒發病率的危險,如新生兒感染、出生后氧療、腦部超聲異常等.阿莫西林加克拉維酸治療與新生兒壞死性小腸結腸炎的發生率明顯增加有關.一個基于1個RCT的系統評價發現,沒有充足的證據證實羊膜腔灌注與不灌注比較能改善胎膜早破后的新生兒結局. (3) 先兆早產的治療:①β-腎上腺素興奮劑:1個系統評價發現,β-腎上腺素興奮劑與安慰劑或不治療相比,并不能明顯降低圍生兒死亡率、呼吸窘迫綜合征及低體重兒(＜2 500 g)發生率,且與與安慰劑或不治療相比,β-腎上腺素興奮劑增加孕母副反應,如胸痛、心悸、呼吸困難、震顫、惡心、嘔吐、頭痛、高血糖、低鉀血癥.②鈣離子通道拮抗劑: 沒有關于鈣離子通道拮抗劑與安慰劑比較的系統評價或RCT.1個系統評價發現,鈣離子通道抑制劑與其它保胎藥(主要是β-腎上腺受體興奮劑)比較,能顯著降低48 h內的早產分娩,減少因孕母副反應退出治療和新生兒發病率.③硫酸鎂:1個系統評價發現,硫酸鎂與安慰劑比較,并不能明顯降低孕36周前的早產率、圍生兒死亡率、呼吸窘迫綜合征的發生率.另一個系統評價發現,硫酸鎂和其他宮縮抑制劑(β-腎上腺素興奮劑、鈣離子通道拮抗劑、前列腺素合成抑制劑、硝化甘油、酒精和葡萄糖注射劑)比較,并不能明顯降低48 h內早產率(盡管結果沒有差異).④垂體受體拮抗劑(阿托西班):1個系統評價納入 2個RCT,對阿托西班和安慰劑治療早產進行比較有不同的結果.較大樣本的RCT發現,阿托西班較安慰劑能延長孕周,但阿托西班增加了孕28周以下的胎兒死亡率.另一個RCT發現,阿托西班增加了48 h內的早產.⑤前列腺素抑制劑(消炎痛):1個系統評價發現,消炎痛與安慰劑比較,能明顯降低孕37周前的48 h和7天的早產率的證據有限.然而,同時發現消炎痛與安慰劑或不治療相比,并不能明顯降低圍生兒死亡率、新生兒呼吸窘迫綜合征、肺支氣管發育不良、壞死性小腸結腸炎、新生兒敗血癥或低體重兒.但這個系統評價樣本太小,尚不能發現有臨床意義的差異. (4) 擇期或非擇期剖宮產對早產婦女治療效果:1個系統評價結果發現,擇期剖宮產較非擇期剖宮產會增加孕母的發病率,卻不能降低新生兒的發病率和死亡率.但尚不能證明此效果是否對新生兒有臨床意義. (5) 改善早產妊娠結局的干預措施:①對早產者采用皮質類固醇:1個系統評價認為,對可能發生早產的婦女使用皮質激素較安慰劑或不處理能明顯降低早產兒出生后呼吸窘迫綜合征、新生兒死亡率和顱內出血的發生.②促甲狀腺激素釋放激素在早產中的運用:1個系統評價發現,在早產的高危婦女中,促甲狀腺激素釋放激素和類固醇激素聯合應用與單用皮質類固醇激素比較,對新生兒結局的影響無明顯差異,但會明顯增加孕母和胎兒的不良反應.③抗生素:1個系統評價發現,抗生素與安慰劑比較,不能延長孕周、降低新生兒死亡率,但可降低孕母感染率.
Objective To evaluate the feasibil ity of intrauterine abdominal wall defect repair of fetal lamb at late pregnancy. Methods Eight healthy pregnant ewes at 110-115 days of gestation (weighing 14-22 kg) were randomly divided into 2 groups. In group A (n=3), the abdominal wall defect of 5 cm × 1 cm was made in the fetal lambs, then was closed by strengthening suture; in group B (n=5), the abdominal wall defect of 5 cm × 2 cm was made in the fetal lambs, then was repairedby 2 layers of biological patches. After the lambs del ivered naturally, the lambs and their wounds were observed; at 10th day after birth, the scars were harvested for biomechanical and histological observations. Results One ewe of group A and 2 ewes of group B aborted, while the others were successfully del ivered. In group A, the abdominal incisions of 2 lambs healed well with a l ine-l ike scar and mild intra-abdominal adhesion, and the scar thickness was 4-5 mm. In group B, the abdominal incisions of 3 lambs did not heal completely with minor intra-abdominal adhesions, and the scar thickness was 3-4 mm. The wound breaking strength was 16, 20 N in group A and 10, 14, and 18 N in group B, respectively. A sl ight scar was seen in group A; skin ulcer and underlying fibrous connective tissue with inflammatory cell infiltration were seen in group B. Conclusion It was feasible to repair the abdominal wall defect of fetal lamb at late pregnancy in uterine. Small abdominal wall defect can be sutured directly; biological patch can be used to repair larger abdominal wall defect.
Objective To explore the osteogenic potential of cervical intervertebral disc fibroblasts in vitro, to investigate the regulatory factors of recombinant human bone morphogenetic protein 2(rhBMP-2) and tumor necrosis factor α(TNF-α) on osteogenic phenotype of fibroblasts and to discuss the condition that facilitates osteogenesis of fibroblasts. Methods Theannulus fibroblasts cell lines of experiment goats were established in vitro and the biologicspecificity was found. According to different medias, 4 groups were included in this experiment: control group, TNF-α group ( 50 U/ml TNF-α), rhBMP-2 group (0.1 μg/ml rhBMP-2) and TNF-α+rhBMP-2 group (50 U/ml TNF-α+0.1 μg/ml rhBMP-2). Thefibroblasts were incubated in the media for about 3 weeks,and then the markers for osteogenic features were investigated by biochemistry, histochemistry observations. Results rhBMP-2 and TNF-α had no effect on the proliferation of fibroblasts from the experiment goats. rhBMP-2 or TNF-α could stimulate fibroblasts to secrete alkaline phosphatase and collagen type Ⅰ. The combined use of rhBMP-2 and TNF-α or the single use of rhBMP-2 could make fibroblasts to secrete osteocalin and the morphological changes of the fibroblasts were very obvious. Histochemical study of the nodules with specific new bone labeler(Alizarin red S) revealed positive reaction, denoting that the nodules produced by the fibroblasts werebone tissues. There was statistically significant difference(Plt;0.05) inALP activity between 3 experimental groups and control group and in secretion of osteocalcin between rhBMP-2 group, TNF-α+rhBMP-2 group and control group. Conclusion The results point out clearly that rhBMP-2 can induce theosteogenic potential of annulus fibroblasts in vitro.
Abstract: The amniotic fluidderived stem cells (AFSC) possess considerable advantageous characteristics including high proliferation potential, easy availability, low immunogenicity and oncogenicity，and accordance with medical ethnics. Moreover, they do not require the sacrifice of human embryos for their isolation and the cells can differentiate into all three kinds of germs. Accordingly，they initiate a new and very promising field in stem cell research and they will be a potential source of stem cells for therapies related to regeneration medicine of cardiovascular diseases. The research about the AFSC utilization in cardiovascular diseases is just started. Though there were some exciting breakthroughs, there still remain many challenges. In the article，we will discuss AFSC characteristics, influence of amniotic fluid harvesting time on stem cells, isolation and purification, emphasizing mainly on the potential of AFSC differentiation into cardiovascular cells, current situation and problems in this field.
【Abstract】 Objective To isolate and culture human amniotic fluid-derived mesenchymal stem cells (HAFMSCs),to investigate a better cryopreservation protocol of HAFMSCs and to observe the biocharacteristics and the multi-potential of HAFMSCs after cryopreservation for the further fundamental researches and cl inical appl ications. Methods HAFMSCswere isolated from the amniotic fluid of pregnant women during the second trimester by the improved two-step method.HAFMSCs were cryopreserved with different cryopreservation protocols (containing different contents of FBS and DMSO atcryoprotectant) in l iquid nitrogen for 12 weeks. The biocharacteristics of the HAFMSCs after cryopreservation were analyzed. The growth characteristics were observed by MTT method and the growth curves were drawn. The surface antigens of HAFMSCs were detected using flow cytometry, including CD29, CD34, CD44, CD45, CD73, and CD90. The adi pogenic and osteogenic differentiation abil ities of HAFMSCs were observed. The mRNA levels of Oct-4 and Nanog of the HAFMSCs were compared between before and after cryopreservations. Results At 12 weeks after cryopreservation, different protocols had different effects on the cell viabil ity; the better formula of cryoprotectant was 50% DMEM, 40% FBS, and 10% DMSO. After cryopreservation, the cells proliferated rapidly and the growth curves showed “S” shape, which was the same as the cells before cryopreservation. Phenotype showed that HAFMSCs were positive for the surface markers CD29, CD44, CD73, and CD90, and negative for CD34 and CD45. After 21 days of adi pogenic differentiation, the l ipid droplets were observed by oil red O staining. After 21 days of osteogenic differentiation, the calcium mineralizations were verified by von Kossa staining. There was no significant difference (P gt; 0.05) in the mRNA levels of Oct-4 and Nanog between before and after cryopreservations. Conclusion HAFMSCs have rapid proliferation and multi-potential in vitro. The cells have high viabil ities and no changes of the biocharacteristics and differentiation potential ities after cryopreservation for 12 weeks. Cryoprotectant containing 50% DMEM, 40% FBS, and 10% DMSO is a better cryopreservation protocol.
Objective To evaluate the influence of PKH26 labeling on the biological function of the goat nucleus pulposus cells and the biological function of seeded cells in nude mice by in vivo imaging techonology. Methods Primary nucleus pulposus cells were isolated by enzymatic digestion from the nucleus pulposus tissue of the 1-year-old goat disc. The nucleus pulposus cells at passage 1 were labeled with PKH26 and the fluorescent intensity was observed under the fluorescence microscopy. The labeled cells were stained with toluidine blue and collagen type II immunocytochemistry. The cells viability and proliferation characteristics were assessed by trypan blue staining and MTT assay, respectively. Real-time fluorescent quantitative PCR was used to detect the gene expressions of collagen types I and II, and aggrecan. The fluorescent intensity and scope of the nucleus pulposus cells-scaffold composite in vivo for 6 weeks after implanting into 5 6-week-old male nude mice were measured by in vivo imaging technology. Results Primary nucleus pulposus cells were ovoid in cell shape, showing cluster growth, and the cells at passage 1 showed chondrocyte-like morphology under the inverted phase contrast microscope. The results of toluidine blue and collagen type II immunocytochemistry staining for nucleus pulposus cells at passage 1 were positive. The fluorescent intensity was even after labeling, and the cell viability was more than 95% before and after PKH26 labeling. There was no significant difference in cell growth curve between before and after labeling (P gt; 0.05). The real-time fluorescent quantitative PCR showed that there was no significant difference in gene expressions of collagen types I and II, and aggrecan between before and after labeling (P gt; 0.05). Strong fluorescence in nucleus pulposus cells-scaffold composite was detected and by in vivo imaging technology. Conclusion The PKH26 labeling has no effect on the activity, proliferation, and cell phenotype gene expression of the nucleus pulposus cells. A combination of PKH26 labeling and in vivo imaging technology can track the biological behavior of the cells in vivo.
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 compare the effects of the denudedfreeze-dried-amniotic-membrane and the denuded freeze-dried bovine corneal stroma when they were explanted to repair the corneal defect of rabbits. Methods The amnia from healthy human placentae were prepared with the method reported by LUO Jingcong, which were freeze-dried and sterilized. The bovine cornea was also denuded by typsin, rinsed, freeze-dried, and sterilized. Twenty Japan rabbits weredivided into group A(the amniontic group) and group B(the bovine-corneal-stroma group) at random. The defect was made, which was 7.5 mm in diameter and 1/3 ofthe thickness of the cornea, and the two kinds of materials were explanted to repair the defect. The vascularization and the changes of the operated eye were observed. The samples were taken at 2, 4 and 8 weeks for histologicalexamination. Results The explanted materials were not melted or excluded. There were visible neovessels in both groups, yet there was no significant difference between them. According to the histological observation, there was severe inflammation in both groups 2 weeks after operation, the fibroblasts were proliferated, and the collagen fibers were disorganized; however,the reactions became milder from 4 weeks after operations,andthe neovessles could be seen in groups A and B; at 8 weeks, the collagen fibers were more organized in groups A and B; however,there was still a small area of disorganized fibers left. Conclusion The two materials can lead to rejection to some extent, and so they need to be improved.
ObjectiveTo investigate the effect of icaritin on the small cell lung cancer cell lines NCI-H446 and its mechanism. MethodsThe NCI-H446 cells at logarithmic growth phase were divided into control and icaritin groups. The cells in the control group were normally treated and cells in the icaritin group were incubated with icaritin (8 μmol/L). Thiazole blue and flow cytometry were used to examine the proliferation and apoptotic changes in the two groups 48 hours after incubation respectively. Gene expression of Janus kinase 2 (JAK2) and signal transducers and activators of transcription 3 (STAT3) were detected by real-time quantitative polymerase chain reaction. The changes of JAK2, STAT3, phospho-JAK2 (p-JAK2), phospho-STAT3 (p-STAT3), Bax and BCL-2 protein were detected by Western blotting. ResultsCompared with the control group, the proliferation rate of NCI-H446 cells in the icaritin group was significantly lower (P<0.05), but the apoptotic rate of NCI-H446 cells in the icaritin group was significantly higher (P<0.05). After the treatment with icaritin, the expression of JAK2 and STAT3 mRNA had no obvious differences. The Western blotting results showed that there was no significant changes in total JAK2, STAT3 protein (P>0.05), but an increasing trend in p-JAK2, p-STAT3 and Bax was observed with the decreasing of BCL-2 (P<0.05). ConclusionIcaritin can inhibit the proliferation and promote the apoptosis of NCI-H446 cells and the effect may be achieved through JAK2/STAT3 signal transduction pathway.
ObjectiveTo evaluate the effects of icariin on autophagy induced by low-concentration of glucocorticoid and exosome production in bone microvascular endothelial cells (BMECs).MethodsBMECs were isolated from femoral heads resected in total hip arthroplasty and then intervened with hydrocortisone of low concentration (0, 0.03, 0.06, 0.10 mg/mL), which were set as groups A, B, C, and D, respectively. On the basis of hydrocortisone intervention, 5×10?5 mol/L of icariin was added to each group (set as groups A1, B1, C1 and D1, respectively). Western blot was used to detect the expressions of microtubule-associated protein 1 light chain 3B (LC3B) and dead bone slice 1 (p62) after 24 hours. Exosomes were extracted from BMECs treated with icariin (intervention group) and without icariin (non-intervention group), and the diameter and concentration of exosomes were evaluated by nanoparticle tracking analysis technique. The total protein content of exosomes was detected by BCA method, and the expressions of proteins carried by exosomes including CD9, CD81, transforming growth factor β1 (TGF-β1), and vascular endothelial growth factor A (VEGFA) were assessed by Western blot. The BMECs were further divided into three groups: BMECs in the experimental group and the control group were co-cultured with exosomes secreted by BMECs treated with or without icariin, respectively; the blank control group was BMECs without exosome intervention. The three groups were treated with hydrocortisone and Western blot was used to detect the expressions of LC3B and p62. The scratching assay was used to detect cell migration ability; angiogenic ability of BMECs was also assessed.ResultsWith the increase of hydrocortisone concentration, the protein expression of LC3B-Ⅱ increased gradually, and the protein expression of p62 decreased gradually (P<0.01). Compared with group with same concentration of hydrocortisone, the protein expression of LC3B-Ⅱ decreased and the protein expression of p62 increased after the administration of icariin (P<0.01). The concentration of exosomes in the intervention group was significantly higher than that in the non-intervention group (t=?10.191, P=0.001); and there was no significant difference in exosome diameter and total protein content between the two groups (P>0.05). CD9 and CD81 proteins were highly expressed in the non-intervention group and the intervention group, and the relative expression ratios of VEGFA/CD9 and TGF-β1/CD9 proteins in the intervention group were significantly higher than those in the non-intervention group (P<0.01). After co-culture of exosomes, the protein expression of p62 increased in blank control group, control group, and experimental group, while the protein expression of LC3B-Ⅱ decreased. There were significant differences among groups (P<0.05). When treated with hydrocortisone for 12 and 24 hours, the scratch closure rate of the control group and experimental group was significantly higher than that of the blank control group (P<0.05), and the scratch closure rate of the experimental group was significantly higher than that of the control group (P<0.05). When treated with hydrocortisone for 4 and 8 hours, the number of lumens, number of sprouting vessels, and length of tubule branches in the experimental group and the control group were significantly greater than those in the blank control group (P<0.05); the length of tubule branches and the number of lumens in the experimental group were significantly greater than those in the control group (P<0.05).ConclusionIcariin and BMECs-derived exosomes can improve the autophagy of BMECs induced by low concentration of glucocorticoid.