With the transformation of marketization of economy and legislation of society in recent 20 years in China,the traditional mechanism in which medical therapy is decided by patients' relatives and doctors has not been able to offer sufficient protection for the legal rights of patients.So the rights of informed consent and decision making have been transferred passively to the patients' side.But in the field of assisted reproduction technique(ART),informed consent has to face some specific and unique problems.Firstly,being fully informed is sharply against the privacy of his(her) spouse as reproduction involved both the couple and their families.Secondly,it is a question that whether a wife can make her freewill choice when she accepts an ART program,because of the pressures coming from her family,the society,and her low social status well.And thirdly,The ART has the characteristics of both clinical therapy and scientific research as it is imperfect and its protocols still need to be improved.Ethicists should focus on thiese challenges.In the aspect of ethics,the regulations established by government and occupational association are the safeguard of the commonweal of society and general rights and benefits,and the ethics review and informed consent offered the final protection for the patients.

Hoxa3-Pax1/Pax9-Eya1-Six1/4 regulatory pathway seems to be operating during forming the bilateral parathyroid/thymus common primordial in early embryonic development.The specification of the parathyroid domain in the parathyroid/thymus primordial is regulated through a Shh-Tbx1-Gcm2 pathway.Gcm2 also may play roles in later steps of parathyroid development,including CaSR and PTH gene expression.MafB and Gcm2 interact with each other and synergistically activate PTH transcription.Genetic basis and the etiology of some hypoparathyroid disorders in man are involved defects in transcription factors that include GCMB,GATA3,Tbxl,SOX3 and GNA11.This marker expression in thymus and parathyroid primordium includes HoxA3,Pax1,Eya1,and Six1;and expression of parathyroid cell-like cells includes Gcm2,CaSR,and PTH.These expressions may serve as markers of stem cell differentiation into parathyroid cell-like cells.

BACKGROUND: Key point for subculture of human embryonic stem cells (hESCs) is to inhibit spontaneous differentiation and make sure totipotency of cells. Although mouse embryonic fibroblasts (MEF) or human foreskin fibroblasts (HFF) used as the feeder layer can maintain undifferentiated state of embryonic stem cells, cell clone is still imperfect and parallel arranged. OBJECTIVE: To establish mixed feeder layer of mouse embryonic fibroblasts plus human foreskin fibroblasts and to observe the hESCs growth.DESIGN: Multi-sample observation and comparison.SETTING: Medical Center of Reproduction, the Affiliated Hospital of Hainan Medical College. MATERIALS: This study was performed at the Medical Center of Reproduction, the Affiliated Hospital of Hainan Medical College from April 2006 to July 2007. Foreskin was derived from the children who underwent circumcision and came from Urinary Surgery, the Affiliated Hospital of Haihan Medical College. The children's family members provided the informed consent, and the experiment received confirmed consent from the local ethic committee. hESCs line HN-1 was separated from human blastula. Eleven 12.5-14.5-day-old fetal mice of clean grade were selected in this study. The experimental animals were disposed according to ethical criteria. METHODS: Heads, four extremities, and viscera were removed from fetal mice under anesthesia. Subsequently, cell suspension was prepared using routine trypsinase digestion and inoculated. When cells were cultured in confluent monolayer, some primary cells were frozen, processed with mitocin-C for 2.0-3.0 hours, and inoculated based on the density of 1×108 L-1 in gelatin-coated dish, I.e., MEF feeder layer. The HFF separation and culture and the preparation of HFF feeder layer were the same as above-mentioned processing. In addition, the two fibroblasts were respectively counted and mixed together according to the ratios of 1∶0, 3∶1, 1∶1, 1∶3, and 0:1. And then, the mixture was inoculated based on the density of 1×108 L-1 in gelatin-coated dish, I.e., mixed feeder layer. The growth of subcultured hESCs in vitro was observed in three different feeder layers, and hESCs in the mixed feeder layer underwent alkaline phosphatase test, OCT-4 expression immunohistochemical measurement, and OCT-4 and telomerase mRNA expression RT-PCR detection. Finally, differentiation in vitro of hESCs was observed after removing the feeder layer.MAIN OUTCOME MEASURES: ① Growth of hESCs in the three different feeder layers; ② Growth of hESCs in the mixed feeder layer based on different mixed ratios; ③ undifferentiated state of hESCs in the mixed feeder layer; ④ differentiation in vitro.RESULTS: ① hESCs clone in the MEF and HFF feeder layers was thin and flat and imperfect, but hESCs clone in the mixed feeder layer was perfect and thick and solid. Apparently, the clone form in the mixed feeder layer was superior to MEF and HFF feeder layers. ② When MEF and HFF was mixed together according to the ratio of 1∶1, hESCs grew in apparent accumulation; clone border was clear; eminentia was apparent and perfect. However, there were no changes according to the ratio of 1∶3. The ratio of 1∶1 was superior to the ratios of 1∶0, 3∶1, and 0∶1. ③ Alkaline phosphatase staining and OCT-4 antigen expression were strongly positive. Specific straps of OCT-4 and telomerase mRNA expression were observed at 200-300 bp and 300-400 bp, respectively. ④ Embryoid bodies were formed. hESCs could differentiate into multi-morphological cells after attachment.CONCLUSION: ① The mixed feeder layer may well support in vitro subculture of hESCs to acquire excellent clone form compared to MEF or HFF feeder layer. ② The mixture of MEF and HFF has excellent effect according to the ratio of 1∶1.

OBJECTIVE To investigate macrolide resistance and main molecular mechanisms in Mycoplasma pneumoniae. METHODS Thirty two throat swabs from children infected with M. pneumoniae were cultured by modified Hayflick medium. Antibiotic susceptibility test was used to screen the macrolide-resistant M. pneumoniae. The 23S rRNA gene sequences of the strains were determined with polymerase chain reaction and sequencing. RESULTS Nineteen strains were isolated from 32 throat swabs successfully.Fifteen strains were resistant to macrolide antibiotics according to the results of antibiotic susceptibility test. Once the strain was resistant to one of macrolide antibiotics,it would be resistant to the others. Sequencing results of the sensitive strains and the standard strain FH were completely same. Fifteen resistant strains presented A2063G point mutation in 23 SrRNA region Ⅴ, in which 2 examples showed the coexistence of the sensitive strain and the resistant strain. CONCLUSIONS Macrolide-resistant M. pneumoniae is common and serious at present. The antibiotic resistant isolate carries point mutations of the 23S rRNA region Ⅴ.

AIM: To investigate the effect of antisense RNA on osteopontin (OPN) expression in renal tubular epithelial cells. METHODS: Cell clone expressing stably OPN antisense RNA was formed by transfering retroviral vector expressing OPN antisense RNA into renal tubular epithelial cells, NRK52E cells, using liposome, with cell clones transfected by empty vector and vector expressing OPN sense RNA as controls. Ribonuclease protection assay(RPA), Western Blot, ELISA and assay of OPN activity were performed to detect expression of OPN mRNA and protein in above clones cultured with or without epidermal growth factor(EGF). RESULTS: The antisense RNA was only expressed by antisense clone. Antisense clone, sense clone and empty clone all expressed OPN mRNA. EGF enhanced expression of OPN mRNA, but not OPN antisense RNA or OPN sense RNA in above clones. OPN protein was not expressed in antisense clone cultured with or without EGF and empty clone cultured without EGF, but was expressed in sense clone cultured with or without EGF and empty clone cultured with EGF. CONCLUSION: Antisense RNA can inhibit OPN protein expression by means of preventing OPN mRNA translation, but not inhibit OPN mRNA transcription in renal tubular epithelial cells.

Objective To investigate the influence of total progressively motile sperm count(TPMSC) after treatment on clinical outcomes of intrauterine insemination(IUI) with the husband′s sperm in ovulation-promoting cycles.Methods The clinical data in 4179 cases undergoing IUI with the husband′s sperm in ovulation-promoting cycles were retrospectively analyzed.The correlation between clinical pregnancy rate and TPMSC was analyzed.Results Among all the clinical data,TPMSC was to 100×106 in occasional live sperm.TPMSC<0.15×106 was in 15 cases,1 case had pregnancy (live sperm was occasionally seen on IUI day after sperm processing).Ten cases of TPMSC >60×106 had no pregnancy.A total of 4 154 cases of TPMSC (0.15-60.00)×106 were analyzed.The female age,duration of infertility,number of follicles and endometrial thickness(EDM) had no statistical differences among various groups.The clinical pregnancy rate was 13.5%(576/4 154),the group with the highest clinical pregnancy rate was (5.00-<10.00)×106.But there was no statistically significant difference in clinical pregnancy rate among groups(P=0.133).Conclusion Performing IUI in PMSC (0.15-60.00)×106 after processing can get preferable pregnancy rates.

Objective To investigate whether TG2 promotes drug resistance to epirubicin through AKT signal pathway in breast cancer. Methods MCF-7 cells with constant expression of TGM2 gene(TGM2-LV)were established via the lentiviral vector. The breast cancer cells were divided into five groups,including the NC group, TG2 group and MK2206 group. The MCF-7/adr cells were divided into ADR group and MKadr group. The expres-sion of TG2 ,AKT ,Bcl-2 and P53 was detected by Western blot assay. Cells were treated with epirubicin. MTT assay was performed to assess cell proliferation. The inhibition ratio of cancer cell proliferation was evaluated. TUNEL analysis was performed to identify the apoptosis of the breast cancer cells. Results Lvels of TG2,p-AKT and Bcl-2 in NC group were significantly lower than those in TG2 group,while the expression of P53 in NC group was much higher. In MK2206(or MK/adr )group,p-AKT and Bcl-2 were down-regulated,while P53 was markedly up-regulated compared with TG2(or ADR)group(P<0.05). The results of the MTT assay showed a strong inhibi-tion in cell proliferation rate in MK2206(or MKadr )group. Compared with the NC group,TG2 promoted prolifera-tion of MCF-7 cells in TG2 group. The cell apoptosis rate in MK2206(or MKadr )group was significantly higher than that in TG2(or ADR)group(P<0.05). TG2 significantly inhibit the apoptosis of breast cancer cells ,com-pared to the control group. Conclusion TG2 might promote drug resistance to epirubicin through AKT signal path-way in breast cancer

Objective To investigate the value of MRI in thmmbolytic thempy of hyperacute cerebral arterial thrombosis.Methods One hundred and sixteen patients with acute cerebral arterial thrombosis were recruited.plain CT and multi.modal MRI were performed in all patients.Thirty-three patients with hyperacute cerebral infarction were treated bv recombinant tissue plasminogen activator(rt-PA) and followed-up periodically using MRI.Results The 33 patients with thrombolysis selected by MRI demonstrated clinical improvement.90 d moclified Rankin scale scores(mRs)were less than 2 and life quality Barthal indexes(BI)were from 80 to 100.The complication included one asymptomatic parenchymal hematoma(PH1)one weeks after thrombolytic therapy and 4(12.2%)hemorrhagic infarction(HI)6 to 24 hours after thrombolytic therapy.Condusions MRI has significant clinical value for the screening and follow-up of intravenous thrombolytic therapy of hyperacute ischemic stroke.MRI-based thrombolysis is a safe and effective method for hyperacute ischemic stroke.

Objective:To develop a questionnaire on knowledge, attitude and practice of Helicobacter pylori (Hp) infection among physical examination population, and to verify its reliability and validity.Methods:This study was a cross-sectional survey. Based on the theory of knowledge, attitude and practice, the first draft of the questionnaire on knowledge, attitude and practice of Hp infection in physical examination population was designed by means of retrospective literature research, qualitative interview, discussion in the research group. The structure and items of the questionnaire were consulted and revised by the expert consultation method (Delphi method). Through pre investigation of 186 physical examination personnel from May to June in 2021, the final version of the "knowledge, attitude and practice questionnaire on Helicobacter pylori infection among physical examination population" was formed after adjustment and verification of the reliability and validity of the questionnaire.Results:The questionnaire of knowledge, attitude and practice of Hp infection in physical examination population included 3 dimensions and 28 items, and the cumulative variance contribution rate was 56.271%. The content validity index of each item level of the questionnaire was 0.75-1.00, and the content validity index of the total questionnaire was 0.94. The Cronbach alpha of knowledge, attitude and practice dimensions in this questionnaire were 0.862, 0.901 and 0.798 respectively. The overall Cronbach alpha of the questionnaire was 0.890, and the half reliability was 0.698. The test-retest reliability of the questionnaire was 0.919, and the test-retest reliability of each dimension was 0.924, 0.917 and 0.845.Conclusions:The questionnaire has good reliability and validity, and can be used to measure the level of knowledge, attitude and practice of Hp infection in physical examination population.

Objective To investigate the epidemiological information of patients in pre-hospital medical care for our large and medium-sized cities and probe the patients' characteristic. Method The data in 2008 were exported from the computer databases of 8 large and medium-sized cities' emergency medical centers in our country.The thorough records of data were conducted to statistical analysis. Results ( 1 ) The scheduling time, running time, rescue time, returning time, total time and service radius in the pre-hospital medical care group were 2.16± 1.10(min), 14.01 ±6.82(min), 12.12±5.96(min), 14.08± 6.85(min), 42.34± 20.21(min)and 8.50±4.18(km), and the above parameter in the non-death group were 2.19 ± 1.13(min), 14.15 ± 7.14(min),11.60±6.72(min), 14.92 ±6.89(min), 41.86± 19.53(minutes) and 8.63±4.31(Km), and the above parameter in the death group were 2.10± 1.08(min), 13.68 ± 7.14(min), 25.25 ± 12.34(min), 13.75±6.48(min), 54.74 ± 25.47(min) and 7.86± 3.91(Km), and the above parameter in the non-sudden cardiac death group were2.09± 1.03(min), 13.58±6.78(min), 25.53± 12.34(min), 13.60± 6.54(min), 53.79±23.77(min) and 7.67 ± 3.86(Km), and the above parameter in the sudden cardiac death group were 2.12 ±1.02(min), 14.10±7.05(min), 24.79± 12.08(min), 13.79±6.61(min), 54. 80 ± 25. 36( min) and 7.90±3.92(Km) respectively. The scheduling time, running time, returning time and service radius in the death group were less than those of the non-death group, but the rescue time and total time of the former were more than those of the latter respectively ( P ＜ 0.05 or P ＜ 0. 001 ). The scheduling time and returning time didn' t have significant difference between the sudden cardiac death group and the non-sudden cardiac death group respectively ( P ＞ 0.05), but the running time, total time and service radius of the sudden cardiac death group were more than those of the non-sudden cardiac death group, and the rescue time of the former was less than that of the latter respectively ( P ＜ 0.05 or P ＜ 0.001 ). (2)The patients' amount in pre-hospital medical care group, the non-death group, the death group, the non-sudden cardiac death group and the sudden cardiac death group were at most in first quarter, and the least time slice of patients' amount were 4:00～ 6:00, 4:00～6:00, 4:00～ 6:00, 22:00～ 24:00, 2:00～4:00 respectively, and the most time slice of patients' amount were 20:00～ 22:00, 20:00～22:00, 8:00～ 10:00, 2:00 ～ 4:00, 8:00 ～ 10:00 respectively. (3)In 241 876 cases of pre-hospital medical care group, the patients' amount of trauma was at most, whose age grades was by far among21 ～50, and the others in sequence were nervous system, circulatory system, other group, digestive system, respiratory system and poisoning group respectively, whose age grades in nervous system, circulatory system and respiratory system was by far above 51, especially above 70. The patients' age grades in other group and digestive system had two climax age groups, which the one was 21 ～ 30, and the other was above 70. The patients' age grades in poisoning group was by far among 21 ～ 50, which the patients' amount of acute alcoholism was at the most. (4) In 12 568 cases of death group, the death amount of circulatory system, other group, respiratory system, nervous system and digestive system ranked at the lst,2nd,4th,5th 8th respectively, whose age grades was by far above 51, especially above 70,and the patients' amount of sudden cardiac death was at the most in the death amount of circulatory system. The death amount of trauma and poisoning group ranked at the 3rd, 6th respectively, whose age grades was by far among 21 ～ 50. (5)The total amount, the death amount and the sudden cardiac death amount of male patients were more than those of female patients. (6)The percentage of the death group to the pre-hospital medical care group was 5.20%, and the percentage of the sudden cardiac death group to the pre-hospital medical care group was 1.29%,and the percentage of the sudden cardiac death group to the death group was 24.87 %, and the percentage of the sudden cardiac death group to the circulatory system group was 67.33 %. Conclusions ( 1 )The trauma and the sudden cardiac death are the overriding reason of disease and the overriding reason of death in our large and medium-sized cities respectively. (2) It is very important to cut the death rate of the middle-old age patients by strengthening prevention and cure of cardiovascular and cerebrovascular diseases, discerning the critical illness early and improving the level of pre-hospital medical care. (3)It is a strong method to decrease the total amount and the death amount of the trauma, especially in traffic accident, by strengthening safety in production, observing traffic regulation and enhancing the legal awareness.