Abstract: Objective To investigate the influence of cryopreservation on cellular viability of latepregnancy fetal valved allografts in human. Methods The fetal valved allografts with gestational ages ranged from 24 to 40 weeks were sterilely procured within 6 hours after brain death. Each sample was bisected into control group and experiment group. The cellular viability of control group was directly tested and that of experiment group was examined after being storaged in liquid nitrogen for a week through a programmed frozen procedure. The light microscopy, tissue culture and Methylthiazol tetrazolium assay (MTT assay) were used to determine the cellular viability. Results Twelve latepregnancy fetal valved aortic allografts were procured. Light microscopy showed the integrity of the basic structure of the thawed aorta, the normal structure of the collagen and elastic fibers, with part of vascular endothelium lost. There were lots of cells deriving from both groups,but the cellular growing rate of the experiment group was relatively slower. At 490 nm, MTT assay valve of control group was 0.442±0.046, and that of experiment group was 0.424±0.041. The difference between two groups failed to statistically significance(t=1.617, P=0.328). Conclusion There were viable cells in latepregnancy fetal valved allografts after cryopreservation.
Abstract In order to repair the bone defect afteroperation of benign lesion of extremity, the fetal demineralized bone was applied in 10 cases. These cases were followed up for 6 months to 8 years. The results showed that the grafted bone was integrated with the host bone in 6 months. Noadverse effect was found. The demineralized bone did not induce rejection. The advantages of using fetal demineralized bone were as follows: easily obtainable,its preparation and method of storage simple, and low finacial cast.
Objective To investigate effects of neural retina on development of the structure of outer blood retinal barrier in embryogenesis. Methods The retinal neural epithelium (RNE) and pigment epithelium (RPE) layers of 150, 120 and 90 embryonic chicken eyes incubated for 7,10, and 14 days were peeled off. RNE was used to prepare the culture medium with different conditions (7drcSF3, 10drcSF3, 14drcSF3). RPE cells of 7- and 14-incubated chicken embryos were cultured on laminin-coated transwell filter. The SF3, 7drcSF3, 10drcSF3 , 14drcSF3 medium were used respectively in the apical chamber and SF2 was used in basolateral chamber. After the formation of monolayer, the transepithelial electrical resistance of the RPE was detected. After the fixation of RPE cells, the condition of the tight junction among the cells was observed by immunohis tochemistry and transmission electron microscopy. Results For the RPE cells of 7-and 14-day incubated embryonic eyes, the difference of TER in various medium of SF3/SF2, 7drcSF3/SF2, 10drcSF3/SF2, 14drcSF3/SF2 was statistically significant (P＜0.01). The polarity of RPE cells was induced and the netlike tight junctional strands was urged in the retina-conditioned medium. Conclusion The neural retina may actively promote the formation of the structure of outer blood retinal barrier. (Chin J Ocul Fundus Dis，2004，20：237-240)
ObjectiveThis study aims to compare different references for the fetal risk of drugs used in pregnancy to provide evidence for the safety of drug use in pregnancy.MethodsFour drug databases, including Lexicomp, Micromedex, TERIS, and Reprotox, as well as two books of drugs in pregnancy edited by Briggs and Schaefer, were searched. Descriptive analysis was performed regarding the definition of pregnancy recommendations and the specific content of medication.ResultsThe six references employed slightly different approaches to drugs in pregnancy, however, all of them included summaries of the risk in pregnancy, data of crossing the placenta, and human and animal data. The databases of Micromedex, TERIS, and a book edited by Briggs had their risk classification systems for drug use during pregnancy. For specific drugs, the summary of different information in pregnancy was different, the amount and content of listed evidence varied, and there was no evaluation of the quality and relevance of evidence among the references.ConclusionsThere is no consensus on the risk assessment of drugs in pregnancy. Risk classification systems for drugs in pregnancy are still an important method for determining the fetal risk of drugs. The existing references merely list studies of drugs in pregnancy, without comprehensive quality assessment. A methodological study of assessment of the risk of drugs in pregnancy is required.
Objective To investigate the feasibility of fetal liver cells for liver tissue engineering, the supporting function of poly L lactic acid (PLLA) scaffold for fetal liver cells and the effects of oncostatin M (OSM), nicotinamide (NA) and dimethyl sulfoxide(DMSO) on growth and hepatic differentiation. Methods After three dimensional PLLA scaffolds having a porous structure were prepared by using NH 4HCO 3 particle, fetal liver cells obtained from E14.5 C57BL/6CrSlc murine embryos were inoculated in the scaffolds. Cells were cultured in Williams’E medium with or without OSM, NA and DMSO for 30 days. Changes in cell number, liver-specific function, and cellular morphology were observed. Results When compared with in monolayer culture, cell number and albumin secretion increased obviously in three-dimensional PLLA. Alburmin secretion increased slightly in OSM group of monolayer culture, but increased obviously in OSM groupo of PLLA culture and in OSM/NA/DMSO group of both monlayer and PLLA cultures. Conclusion The three-dimensional PLLA scaffold is a good supporting material for the cultivation of tetal liver cells. OSM, NA and DMSO remarkaly stimulated maturation of hepatic parenchymal cells in vitro in terms of morphology and liver-specific function.
【摘要】 目的 观察胎羊宫内心脏介入手术胎羊血气及血浆炎性细胞因子的变化。方法 8只怀孕双胎山羊,双胎之一为实验组,在相同麻醉条件下,实验组进行胎羊心脏介入治疗,并抽取血样标本。监测胎羊的心率、血气、乳酸值,运用ELISA法检测治疗组及对照组胎羊白介素(IL)1、IL6、IL8及肿瘤坏死因子(TNFα)。结果 2只胎羊因手术中发生心包填塞死亡,存活的6只胎羊手术前pH值较手术后有明显下降(Plt;005),手术前后乳酸浓度上升(Plt;005),PCO2、PO2差异无统计学意义(Pgt;005),手术前血浆IL1、IL6、IL8的浓度较手术后高(Plt;005),手术前后TNFα的浓度变化无统计学意义(Pgt;005)。结论 胎羊宫内心脏介入手术可引起胎羊血浆pH值下降,乳酸浓度上升,及细胞因子IL1、IL6、IL8浓度上升。【Abstract】 Objective To observe the change of blood gas and inflammatory cytokines during intrauterine cardiac intervention surgery on the fetal lambs. Methods Eight pregnant goats with two fetal in each goat were included. With the same anesthesia condition, one of the twin fetus was chose to perform the intrauterine cardiac intervention surgery. The fetal heart beating rate was monitored, and blood samples of the fetus were taken to do the blood gas analysis and to detect the concentration of inflammatory cytokines (IL1, IL6, IL8, and TNFα). Results Two of the eight fetal lambs which was died in the operation because of pericardial tapenade. In the other six survived fetus, the PH was lower than after the surgery, and the concentrations of lactic acid, IL1, IL6, and IL8 are higher than after the surgery. There was no significant difference of PCO2,PO2 and TNFα between before and after the surgery. Conclusion The intrauterine cardiac intervention surgery can make the PH of fetal plasma lower and the concentrations of lactic acid and IL1, IL6, IL8 higher.
Objective To explore the change tendency of hypoxia-inducible factor-1α (HIF-1α) and extracellular signal-regulated kinase 1/2 (ERK1/2) in fetal rat cerebral cortex neurons cultured in vitro after hypoxia-ischemia reperfusion andto investigate their mutual relationship. Methods Cortical neurons obtained from cerebral cortex of 15 pregnant SD rats at16-18 days of gestation underwent primary culture. The primary neurons 5 days after culture were adopted to establ ish model of oxygen and glucose deprivation (OGD). The experiment was divided into 4 groups: the experimental group 1, culture medium was changed to neuron complete medium containing glucose after the preparation of OGD model to form reperfusion, and the neurons were observed 0, 2, 4, 8, 12 and 24 hours after reperfusion; the control group 1, the neurons were treated with normal medium; the experimental group 2, the neurons were pretreated with U0126 followed by the preparation of OGD model, and the neurons were observed 4 and 8 hours after reperfusion; the control group 2, the neurons were pretreated with DMSO, and other treatments were the same as the experimental group 2. Expressions of HIF-1α, VEGF protein, ERK1/2 and p-ERK1/2 were detected by Western blot. Expression and distribution of p-ERK1/2 and HIF-1α protein were detected by SABC immunocytochemistry method. Results Compl icated synaptic connections between cortical neurons processes were observed 5 days after culture. The expression of HIF-1α and VEGF were increased gradually, peaked at 8 hours, and decreased gradually after 12 hours in the experimental group 1, and there were significant differences between the experimental group 1 and the control group 1 (P lt; 0.05). There was no significant difference between the experimental group 1 and the control group 1 in terms of ERK1/2 protein expression (P gt; 0.05). The p-ERK1/2 protein expression in the experimental group 1 started to increase at 2 hours peaked at 4 hours, and started to decrease at 8 hours, showing significant differences compared with the control group 1 (P lt; 0.01). In the experimental group 2, the p-ERK1/2 protein decreased, and HIF-1αand VEGF protein expression subsequentlydecreased, showing significant differences compared with the control group 2 (P lt; 0.05). There was no significant difference between the experimental group 2 and the control group 2 in terms of ERK1/2 protein expression at each time point (P gt; 0.05). Immunocytochemistry staining showed that p-ERK1/2 and HIF-1α expression decreased, and the yellow-brown staining of the neurons was reduced. Conclusion Expressions of HIF-1α and its target-gene VEGF protein in the cortex neurons after OGD reperfusion are time-dependent. Their expressions decrease when ERK1/2 signal ing pathway is inhibited, indicating the pathway plays an important role in the regulation of HIF-1α and VEGF induced by OGD of cortical neurons
OBJECTIVE: To explore the effects of different methods of fetal spinal cord(FSC) tissue transplanted on reversing the axotomy-induced neurons atrophy of adult rats injured spinal cord. METHODS: One hundred and twenty adult rats received lumbar spinal cord hemisection. Experimental rats were divided into five groups, the control group(Group A); spinal cord hemisection only(Group B); spinal cord hemisection plus FSC transplant (Group C); spinal cord hemisection plus FSC transplant plus pedicled paraspinal muscle(Group D); spinal cord hemisection plus FSC transplant plus pedicled omentum (Group E). Combined behavioral scores(CBS), somatosensory evoked potentials (SEP), motor evoked potentials(MEP) were examined to evaluate the recovery of neurological function after operation. Rats were sacrificed after 1, 4 and 12 weeks. Nissl stained section was used for neurons quantitative image analysis. The positive cells were quantitative analysis by computer image analysis system. RESULTS: The different methods of FSC tissue transplantation could prevent the neurons atrophy secondary to axon injury of spinal cord in adult rats. The size of neurons were observed in five groups, they were group E gt; group D gt; group C gt; group B gt; group A (P lt; 0.05). Those increases in size of neurons were paralleled with a significant improvement in neurological function recovery. CONCLUSION: It indicates that the different methods of FSC tissue transplantation can maintain the neurons morphology and improve the neurological function of rats.
Objective To investigate the possibility of culturing human oral keratinocyte using autologous serum in order to provide theoretical and technical foundation for clinical application of tissue engineering oral mucosa epithelium.Methods The human oral keratinocytes were cultured by the medium containing different concentrations of autologous serum(10%,20%,30%)and fetalbovine serum (10%), respectively. The growth conditions for the cell and the mucosa epithelium in the groups were observed, the cell growth curves were drawn, and the population doubling time (PDT) was counted. Results The results showed that the human oral keratinocyte could proliferate well in the medium containing autologous serum or fetal bovine serum. The differences in the 24hour clone rate and PDT were not significant. Both the area and the thickness of the cultured oral epithelium increased with the increase of the autologous serum concentration, and the difference between autologous serum and fetal bovine serum was significant, especially with the medium containing 20% autologous serum( P＜0.05) . The human nature of the cultured epithelium was demonstrated by the immunofluorescent mouse anti-HLA antigen. Conclusion The autologous serum can replace the fetal bovine serum to culture the oral keratinocyte well, and the cultured oral mucosa epithelium can be better differentiated in the autologous serum than in the fetal bovine serum.
ObjectiveTo explore the effect of fetal bovine serum (FBS) of different concentrations in the culture medium on osteogenic growth peptide (OGP) promoting bone marrow mesenchymal stem cells (BMSCs) proliferation and differentiation. MethodsBMSCs were separated from limb bones of 8 Sprague Dawley rats (5 weeks old) and purified by adherence method, and BMSCs at passage 3 were divided into 4 groups according to OGP concentration: OGP 1×10-10 mol/L group, OGP 1×10-9mol/L group, OGP 1×10-8 mol/L group, and control group without OGP; and 0, 2%, 5%, 8%, and 10%FBS concentration gradient was used in each group. The cell proliferation rate was detected by MTT method at 1, 3, 5, 7, 9, and 12 days after culture, and the activity of intracellular alkaline phosphatase (ALP) was determined by the method of p-nitrophenyl phosphate disodium at 9 days after culture. ResultsBMSCs showed adherent growth, rapid proliferation, long fiber vortex, and typical morphology. MTT analysis showed that cells could not sustain proliferation when FBS concentration was less than 5% in each group; when FBS concentration was above 8%, cells proliferated continually. Proliferation promoting effect of OGP 1×10-8 mol/L and 1×10-9 mol/L groups was significantly higher than that of the control group in all serum concentrations (P<0.05); when FBS concentration was lower than 10%, the proliferation promoting effect of OGP 1×10-8 mol/L group was significantly higher than that of the other 2 OGP groups (P<0.05), but when FBS concentration was 10%, OGP 1×10-8 mol/L group had no advantage of promoting proliferation. ALP test results showed that as the FBS concentration increased, ALP activity of all groups also significantly increased (P<0.05). Under the condition of 5%FBS and 8%FBS, the ALP activity of each OGP group was significantly greater than that of the control group, and it was the highest in OGP 1×10-8 mol/L group (P<0.05). Under the condition of 10%FBS, the ALP activity of each OGP group was still greater than that of the control group (P<0.05), but no significant difference was found between the OGP 1×10-8 mol/L group and OGP 1×10-9 mol/L group (P>0.05). ConclusionThe concentration of 8%FBS is the best concentration of serum for OGP promoting the proliferation and differentiation of BMSCs, and the most suitable concentration of promoting the proliferation and differentiation of BMSCs is OGP 1×10-8 mol/L.