【Abstract】 Objective To investigate the effect of IGF-1 on the growth of primary human embryonicmyoblasts. Methods The method of incorporation of 3H-TdR was used to evaluate the abil ity of prol iferation of myoblasts.The count per minute (CPM) values of myoblasts at different concentrations(1, 2, 4, 8, 16 and 32 ng/mL) of IGF-1 were measured,and dose-effect curves were drawn to choose the optional concentration of IGF-1 to promote the prol iferation. Then theexperimental group of myoblasts received the addition of the optional concentration of IGF-1 in the growth medium, the controlgroup just received the growth medium. The flow cytometry was used to detect the cell cycle . The method of incorporation of3H-TdR was used to measure the peak-CPM. The myotube fusion rate was measured in myoblasts with different concentrations(0, 5,10, 15, 20, 25 and 30 ng/ mL) of IGF-1 in fusion medium, the dose-effect curves were also drawn, so as to decided the optional concentrationof IGF-1 in stimulating differentiation. Fusion medium with optional concentration of IGF-1 was used in experimentalgroup, and the control group just with fusion medium. The fusion rate of myotube and the synthesis of creatine kinase(CK) weredetected in both groups. Results The optional concentration of 5 ng/mL IGF-1 was chosen for stimulating prol iferation . It was shown that the time of cell cycle of control was 96 hours, but that of the experimental group was reduced to 60 hours. The results of flow cytometry showed that the time of G1 phase, S phase and G2M phase was 70.03, 25.01 and 0.96 hoursrespectively in control group, and were 22.66, 16.47 and 20.87 hours respectively in experimental group. The time-CPM value curves showed that the peak-CPM emerged at 96 hours in control group and 48 hours in experimental group, which was in agreementwith the results of the flow cytometry. The optional concentration stimulating prol iferation was 20 ng/mL IGF-1. Compared with control, the quantity of CK was increased by 2 000 mU/mL and the fusion rate was elevated by 30% in experimental group. Conclusion The concentrations of 20 ng/mL IGF-1 can elevat obviously the fusion rate and the quantity of CK. IGF-1 can enhance the prol iferation and differentiation of myoblasts via inducing the number of myoblasts at G1 phase and increasing the number of myoblasts at S and G2M phases.
Human SW480 colonic cancer cell line was evaluated for its growth response to Octa peptide somatostatin (SMS 201·995, SMS) in vitro by MTT assay and flow cytometry. The results showed that SMS possessed an inhibitive effect on SW480 cell at dose 1.563－200ng/ml, the maximal effective dose was 50ng/ml. Inhibitive effect of SMS did not steadily increase at a dose ＞50ng/ml. It suggests that effect of SMS is achieved via somatotatin receptor. SMS obviously inhibited the synthesis of DNA and protein, and prohibited the SW480 cell shifting from phase G0/G1 in phase S, G2M, which suggests that somatostatin (SS) possessed an inhibitive effect on large intestinal at cancer cell, it is achieved at receptor by inhibiting the synthesis of DNA and protein and prohibiting cell cycle of cancer.
This study aimed to investigate the effect of curcumin (Cur) against human cytomegalovirus (HCMV) in vitro. Human embryonic lung fibroblasts were cultured in vitro. The tetrazolium salt (MTS) method was used to detect the effects of Cur on cell viability. The cells were divided into control group, HCMV group, HCMV + (PFA) group and HCMV + Cur group in this study. The cytopathic effect (CPE) of each group was observed by plaque test, then the copy number of HCMV DNA in each group was detected by quantitative polymerase chain reaction (qPCR), and the expression of HCMV proteins in different sequence was detected by Western blot. The results showed that when the concentration of Cur was not higher than 15 μmol/L, there was no significant change in cell growth and viability in the Cur group compared with the control group (P>0.05). After the cells were infected by HCMV for 5 d, the cells began to show CPE, and the number of plaques increased with time. Pretreatment with Cur significantly reduced CPE in a dose-dependent manner. After the cells were infected by HCMV, the DNA copy number and protein expression gradually increased in a time-dependent manner. Pretreatment with Cur significantly inhibited HCMV DNA copies and downregulate HCMV protein expression levels in a concentration-dependent manner, and the difference was statistically significant (P<0.05). In conclusion, Cur may exert anti-HCMV activity by inhibiting the replication of HCMV DNA and down-regulating the expression levels of different sequence proteins of HCMV. This study provides a new experimental basis for the development of anti-HCMV infectious drugs.
Objective To investigate the role of anti apoptosis gene bcl-2 in the survival of cultured uveal melanoma UM cells. Methods Antisense oligodeoxynucleotides (AS-ODN) bcl-2 were delivered with cationic lipid to primary cultured UM cells. The inhibitory effect of AS-ODN bcl-2 on proliferation of UM cells was examined by 3,-4,5 Dimethyliazol-2,5 diphenyl tetrazolium bromide (MTT) method. Using DNA ladder to determine if the UM cells had been apoptotic. Bcl-2 expression was detected by RT-PCR and Westernblot technics. Results The effect of AS-ODN bcl-2 in inhibiting the proliferation of cultured UM cells had opposite relation to dosage. It down regulated the mRNA and protein level of bcl-2 gene, and the sense ODN didn′t have this effect. Conclusion AS-ODN bcl-2 can down regulate bcl-2 expression, inhibits UM cells proliferation and induces apoptosis. (Chin J Ocul Fundus Dis, 2002, 18: 38-41)
Objective To find a feasible method that can reconstruct the composite tissue-engineered skin fast in vitro and can provide enoughskin as soon as possible for covering the surface of the large-area burn. Methods The foreskin was taken during the posthectomy. The epidermal cells and fibroblasts were isolated, identified and cultured. The cytokeratin 19 (K19) flow cytometry and the fluorescein isothiocyanate (FITC)immunofluorescence for K19 and the FITCimmunofluorescence for PAN-cytokeratin of the epidermal cells and the FITCimmunofluorescence for vimentin of the fibroblasts were performed to identify the epidermal cells and the fibroblasts. Then, the epidermal cells were seeded onto the papillary surface of an acellular dermal matrix (ADM) and were submerged into the condition culture medium added with 25 ng/ml of the keratinocyte growth factor (KGF). However, in the control group, no KGFwas added. After 24 hours, the ADM was moved up to the airfluid surface, and the culture was continued. After 6 days, the fibroblasts were seeded onto the other surface of the ADM. After a 24 hour culture, the ADM was harvested and fixed in formalin, and the hematoxylin-eosin staining was conducted. Then, the structure of the reconstructed skin was observed under the microscope and the cell count in the epidermal layer was also conducted. Results All the cultured and expanded epidermal cells stained by the immunofluorescence demonstrated a positive reaction for PANFITC, and a partially positive for K19-FITC, and 17% of the cells demonstrated a positive reaction for K19 identified by the flow cytometry. The fibroblasts could be expanded by more than 100 times after a 7day culture in vitro, and they could demonstrate a positive reaction for vimentin-FITC. After a 7day culture, a composite tissue-engineered skin could be attained. The hematoxylineosin staining of the reconstructed skin showed that there was one continuous layer of the epidermis on the papillary surface of the ADM and there were fibroblasts in the superficial layer of the other one, but the epidermal layer did not stick tightly to the ADM. The cell count demonstrated that KGF promoted the epidermal cells to proliferate better（Plt;0.01）and to form more significantly continuous layers of the epidermis in the experimental group than in the control group（Plt;0.01）. Conclusion Through the seed-cell separation by the digestion of collagenase and trypsin combined with the use of the KGF-added condition ulture medium, a composite tissueengineered skin can be reconstructed within 7 days.
ObjectiveTo explore the construction of heart preservation model of empty beating donor based on extracorporeal membrane oxygenation (ECMO). MethodsFrom January 2022 to August 2023, 20 Guangxi Bama miniature pigs weighing 25-30 kg were selected, half male and half female. Under general anesthesia and heparinization, a midline thoracotomy was performed. The pericardium was cut after freeing the anterior and posterior vena cavae, and a perfusion needle was inserted near the brachiocephalic artery in the ascending aorta, connected to a blood collection bag to collect 500-600 mL of blood. The anterior and posterior vena cavae were ligated, the aorta was blocked and perfused with HTK solution to stop the heart beating. The superior and inferior vena cavae were cut off, the right pulmonary vein was decompressed, the aorta and left and right pulmonary arteries and veins were cut off, and the whole heart was removed. An ECMO device was used to continuously perfuse a cardioprotective solution mainly composed of oxygenated warm blood, maintaining the isolated pig heart beating for 8 hours, monitoring (once/hour) ECMO perfusion parameters, blood gas indicators, perfusate electrolytes, detecting inflammatory factors, myocardial enzymes, myoglobin, and troponin levels. Myocardial tissue was taken for hematoxylin-eosin (HE) staining to observe myocardial cell damage and evaluate the quality of heart preservation. ResultsAmong the 20 isolated beating preservation pig hearts, 17 successfully resumed beating, 3 experienced ventricular fibrillation, resuscitated after intracardiac electrical defibrillation, and all 20 pig hearts successfully beat for 8 hours. There was no statistical difference in ECMO perfusion parameters, blood gas indicators, perfusate electrolytes, and inflammatory factors at each time point (P>0.05). There were statistical increases in myocardial enzymes, myoglobin, and troponin levels (P<0.05). HE staining results suggested that there was no severe myocardial damage. ConclusionECMO technology can be used for pig heart preservation with good results, and this study provides experimental evidence for improving heart preservation research in clinical heart transplantation.
Objective To evaluate the effectiveness of GnRH antagonist in vitro fertilization-embryo transfer (IVF-ET) in polycystic ovary syndrome (PCOS) patients.Methods Such databases as PubMed (1997 to 2010), PML (1997 to 2010), FMJS (2000 to 2010), CBMdisc (1979 to 2010), CNKI (1994 to 2010), VIP (1989 to 2010), WanFang (1994 to 2010), and duxiu scholar searcher (www.duxiu.com), and nine relevant Chinese journals were searched for retrieving the randomized controlled trails (RCTs) on the effectiveness of GnRH antagonist versus GnRH agonist for IVF-ET in PCOS Patients. The studies were screened according to the inclusive and exclusive criteria by two reviewers independently, the data was abstracted and the quality was evaluated. The RevMan 4.2.7 software was used for Meta-analyses. Results Six grade-B studies involving 699 participants were included. The results of Meta-analyses showed that, compared with the GnRH agonist, there was no significant difference in the GnRH antagonist group about the stimulation duration (WMD= –1.23, 95%CI –2.76 to –0.31), dose of gonadotrophins (Gns) (WMD= –4.87, 95%CI –14.20 to 4.46), serum E2 value on the day of HCG administration (WMD= 31.37, 95%CI –263.40 to 326), number of oocytes retrieved (WMD= 1.34, 95%CI –1.02 to 4.70), clinical pregnancy rate (OR= 1.27, 95%CI 0.77 to2.10), and miscarraige rate (Peto OR= 0.67, 95%CI 0.38 to1.18). But the OHSS rate in the GnRH antagonist group was lower with a significant difference (Peto OR= 0.35, 95%CI 0.24 to 0.50). Conclusions Compared with the GnRH agonist protocol, the GnRH antagonist protocol can obviously reduce the incidence of OHSS, but has the same effect in Gn dose, retrieving oocytes and clinical pregnancy rate. Because the GnRH antagonist can decrease the treatment duration and cost, and has better safety, so it may be an ideal choice for PCOS patients to have IVF-ET therapy. For the quality and quantity limitation, and the methodology difference of the included studies, it is suggested that the conclusion from this study should be only served as a reference of clinical analyses, and should be revaluated and updated unceasingly.
ObjectiveTo observe the protective effect of etomidate (ET) on cultured retinal ganglion cells (RGC) with mechanical injury in vitro. MethodsNew Sprague-Dawley rat RGC was cultured in vitro and identified by double immunofluorescent labeling of Thy1.1 and microtubule associated protein 2. The cultured primary cells were randomly divided into control group, RGC scratch group, ET low dose group (1 μmol/L), ET medium dose group (5 μmol/L) and ET high dose group (10 μmol/L). The RGC mechanical injury model was established by using iris knife to culture cells in RGC scratch group and ET group with different concentration. Seven days after modeling, the RGC survival rate of each group was detected by cell count Kit 8 proliferation assay. The apoptosis rate of RGC was detected by Annexin Ⅴ/propyl iodide double staining. Single factor analysis of variance was used to compare the groups. The pairwise comparison between groups was tested by the least significant difference method. ResultsThe survival rates of RGC in RGC scratch group, ET low dose group, ET medium dose group and ET high dose group were (72.60±2.97)%, (73.73±1.14)%, (79.19±1.79)% and (83.88±0.94)%, respectively. The RGC apoptosis rates of control group, RGC scratch group, ET low dose group, ET medium dose group and ET high dose group were (5.08±0.17)%, (18.67±1.24)%, (17.96±0.74)%, (15.11± 0.56)% and (11.67±1.32)%, respectively. Comparison of RGC survival rate between groups: compared with RGC scratch group, the cell survival rate of ET low-dose group, ET medium-dose group and ET high-dose group was increased, and the difference between RGC scratch group and ET low-dose group was not statistically significant (P=0.728); the differences between RGC scratch group, ET medium dose group and ET high dose group were statistically significant (P＜0.001); the difference between ET medium dose group and ET high dose group was statistically significant (P=0.002). Comparison of apoptosis rate of RGC among groups: the apoptosis rate of RGC scratch group was significantly higher than that of control group, the difference was statistically significant (P＜0.001). Compared with RGC scratch group, the apoptosis rate of ET low-dose group, ET medium-dose group and ET high-dose group was decreased, and there was no statistical significance between RGC scratch group and ET low-dose group (P=0.869). The differences of apoptosis rate between RGC scratch group, ET medium dose group and ET high dose group were statistically significant (P＜0.05). The difference of apoptosis rate between ET medium dose group and ET high dose group was statistically significant (P=0.007). ConclusionET has neuroprotective effect on RGC cultured in vitro with mechanical injury, and the protective effect increases with the increase of ET dose in a certain range.
Objective Titania and Ag containing nano-hydroxyapatite/polyamide 66 (TiO2-Ag-nHA/PA66) composite bone fill ing material has good biocompatibil ity and biological safety. To investigate the antibacterial effect and Ag+ release characteristics of TiO2-Ag-nHA/PA66 composite bone fill ing material containing different concentrations of Ag+ in vitro. Methods The n-HA/PA66 composite bone fill ing material A1 (material A1) was prepared by co-polymerization method, and TiO2-Ag-nHA/PA66 composite bone fill ing materials A2 and A3 (materials A2 and A3) were prepared by thesame way containing Ag+ of 0.22wt% and 0.64wt%, respectively, and the TiO2 content was 2.35wt%. The materials A2 and A3 were respectively immersed in 50 mL simulated body fluid (SBF), and Ag+ concentration was measured by atomic absorption spectrometry at 1, 3, 7, 14, 21, and 49 days. The inhibition ring test and colony count method were used to evaluate antibiotic effect against Staphylococcus aureus and Escherichia coli, the anti-adhesion capacity of Staphylococcus aureus and Escherichia coli was observed by scanning electron microscope (SEM). Results There was no significant difference in the Ag+ concentration between materials A2 and A3 at 1 day and 3 days (P gt; 0.05); and there were significant differences in the Ag+ concentration between materials A2 and A3 after 7 days (P lt; 0.05). The inhibition ring diameters of materials A2 and A3 to Staphylococcus aureus and Escherichia coli reached the maximum at 1 day, which were (13.40 ± 2.88), (9.40 ± 1.14) mm and (23.60 ± 1.14), (18.80 ± 0.84) mm, showing significant difference (P lt; 0.05) between materials A2 and A3 respectively; and then, the diameter of inhibition ring reduced with the time. The antibacterial effect of materials A2 and A3 against Staphylococcus aureus and Escherichia coli lasted 15, 33 days and 9, 24 days, respectively. No inhibition ring was observed around material A1 all the time. And the inhibitory rates of materials A2 and A3 were 89.74% ± 3.62%, 94.18% ± 2.05% and 78.65% ± 5.64%, 85.96% ± 2.50%; showing significant differences (P lt; 0.05) among materials A1, A2, and A3. SEM showed that bacterial adhesion of materials A2 and A3 was obviously fewer than that of material A1. Conclusion TiO2-Ag-nHA/PA66 composite bone fill ing material has antibacterial property against Staphylococcus aureus and Escherichia coli, and it has a good release effect in SBF.
In order to meet the rapid development of in-vitro diagnostic reagent (IVD) market and the development needs of laboratory medicine, and ensure efficient management and cost control in IVD purchasing, it is necessary to build the purchasing management standard, improve the purchasing quality of IVD, reduce the overall hospital costs, and ensure the accuracy and reliability of clinical test results. Through three aspects: the preparation before business negotiation, business negotiation, and execution after business negotiation, this article clarifies the self-bidding procurement process of IVD, and emphasizes the importance of knowledge in IVD procurement management, so as to provide references and suggestions for novice buyers to get familiar with the business quickly and improve their negotiation ability.