Objective To obtain recombinant human granulysin using prokaryotic expression system.MethodsTotal RNA was extracted from cultured PBMC. Granulysin gene segments were obtained with granulysin-specific primers by RT-PCR and then inserted into pET32a(+) plasmid. After identification by DNA sequence,pET-GNLY9K and pET-GNLY15K were transferred to E. coli Rosetta (DE3). The fusion protein was identified by SDS-PAGE and Western blot.The bioactivity of granulysin fusion protein was measured by MTT assay.Results The prokaryotic expression vectors pET-GNLY9K and pET-GNLY15K were successfully constructed.The corresponding protein was highly expressed in E.coli. Recombinant protein was specifically bound by anti-granulysin antibody. GNLY9K fusion protein significantly inhibited the growth of A549 cells in a dose-dependent manner,while GNLY15K had little effect on the growth of A549.Conclusion Granulysins with different mw were successfully expressed using prokaryotic expression system,which might be helpful for the further study of granulysin.

Objective:To investigate the significance of 25 ( OH )-vitamin D in its diagnostic value of primary biliary cirrhosis. Methods:44 cases with PBC,41 cases with Hepatitis B cirrhosis and 110 normal people as control group were all analyzed bi-ochemical results and significance of serum 25 ( OH )-vitamin D. We Used receiver operator characteristic curve ( ROC ) to analyze serum 25(OH)-vitamin D in its diagnostic value of primary biliary cirrhosis. Results:Biochemical results of TBIL,DBIL,IBIL,GLB, AST,ALT,ALP,γ-GT were higher in PBC than that in normal people and Hepatitis B cirrhosis. While the serum 25(OH)-vitamin D of PBC was much more lower than that in normal and people and Hepatitis B cirrhosis. The difference between them were statistical difference(P<0. 05). The area under the ROC curve of PBC was 0. 926,it showed serum 25(OH)-vitamin D diagnosis for PBC had quite value. When the cut-off level of serum 25(OH)-vitamin D was 17. 275 ng/ml,the sensitivity for the diagnosis was 92. 8%,the re-spectively was 84. 1%. Conclusion:Serum 25(OH)-vitamin D diagnosis for PBC has quite value,the cut-off level of serum 25(OH)-vitamin D is 17. 275 ng/ml.

Objective To obtain recombinant human granulysin using prokaryotic expression system. Methods Total RNA was extracted from cultured PBMC. Granulysin gene segments were obtained with granulysin-specific primers by RT-PCR and then inserted into pET32a(+) plasmid. After identification by DNA sequence, pET-GN-LY9K and pET-GNLY15K were transferred to E. Coli Rosetta (DE3). The fusion protein was identified by SDS-PAGE and Western blot. The bioactivity of granulysin fusion protein was measured by MTT assay. Results The prokaryotic expression vectors pET-GNLY9K and pET-GNLY15K were successfully constructed. The corresponding protein was highly expressed in E. Coli. Recombinant protein was specifically bound by anti-granulysin antibody. GNLY9K fusion protein significantly inhibited the growth of A549 cells in a dose-dependent manner, while GN-LY15K had little effect on the growth of A549. Conclusion Granulysins with different mw were successfully expressed using prokaryotic expression system, which might be helpful for the further study of granulysin.

Objective:To investigate the medical students' cognition of ethical issues in assisted reproductive technology (ART) and discuss the importance of ethical education in medical colleges and universities.Methods:A questionnaire survey about the ethical cognition of assisted reproductive technology was conducted among 632 medical students.The questionnaire included the donation and management of gametes and embryos,and offspring problems.Results:Medical students agreed with most of the current ART-related laws and regulations.however,44.8％ of medical students considered that criteria of donating eggs should be relaxed,such as allowing women who do not receive ART treatment be paid for eggs.In addition,a total of 39.0％ of medical students supported the legalization of surrogacy technology in china.Conclusion:Medical students exists a certain misunderstanding in egg donation and surrogacy technology.We should give more training to medical students in ethical knowledge.In addition,our country can supplement and perfect the current laws and regulations to a certain extent.

Objective Analyzes the condition of serum 25-hydroxy Vitamin D (25OHD)level of partial middle and old age physical examinations in Yinchuan area.Methods Tested the level of serum 25OHD of 1 520 cases of senile medical with electrochemiluminescence immunoassay (ECLIA)in October 2013 to September 2014 in Yinchuan,NingXia.exclusion of liv-er,kidney,cancer,diabetes and other diseases,and statistical analysis of each age group and one year 25-(OH)D distribution. Compared gender and 50~59,60~69,>70 years at different ages and spring,summer,autumn,winter four seasons 25OHD level distribution.Results All the mean level was 14.67±8.26 ng/ml.The lack and deficiency of Vitamin D account was 80.66%,and the female was 84.49% significantly higher than the male 74.96% (t=2.03,P<0.05).Used divide subjects into fore groups:the lack (42.76%),the deficiency (37.90%),nomal (13.68%)and good (5.66%)by the level of serum 25OHD.Vitamin D nutritional status was different in person of different ages.Fuither linear correlation analys showedgative correlation between the level of serum 25OHD and age (r=0.16,P<0.01).There was seasonal variation in the level of ser-um 25OHD (F=19.30,P<0.001).It was highest in summer,and lower in spring and winter.Conclusion Vitamin D of middle-aged and old people in Yinchuan was general deficiency,which should pay more attention and actively treatment to these patients.

Objective To investigate the impact of synthetic urine (synurine solution) at differentpH values on skin surface.Methods Sixty-four healthy volunteers were enrolled into this study.Based on the results of lactic acid test and questionaires,these subjects were enrolled as sensitive skin group and normalskin group.The 4-,20-,and 24-hour occlusive patch tests were successively performed with synthetic urine at various pH values (2.0,3.5,5.0,6.5,8.0 and 10.0)on the two groups of volunteers.The distilled waterand 0.25%sodium lauryl sulphate (SLS)served as control.The skin surface pH values were measured bypotable pH meter before,and at 24 h,48 h,72 h after the first patch.Half an hour after the latter three detections of pH values,the skin response was evaluated clinically,as well as by transepidermal water loss (TEWL)and pH values,which were measured by pH-900 pH Meter.Results The responses to these solutions were similar between sensitive and normal skin groups.The order of skin surface pH values measured by the two pH meters was consistent with that of the original solutions.The skin surface pH value was altered by the synthetic urine,and this change was still present at 24 hours after the patches were removed.The TEWL values of skin challenged by synthetic urine were not higher than those by distilled water.The skin responses at 72 hours were more intense to the synthetic urines with pH values of.5,8.0,10.0than to distilled water.CondusionsSynthetic urines with different pH values can alter skin surface pH values,and skin responses to synthetic urines with a pH range 2.0-10.0 are similar to those to distilled water.

Objective To explore the high-risk factors of metabolic bone disease (MBD) in premature infants by retrospective analysis of the clinical data so as to provide evidence for optimal clinical management. Methods Clinical data of premature infants with birth weight<1500 g admitted in our hospital from January 2016 to December 2017 were retrospectively analyzed. Infants with serum alkaline phosphatase ( ALP )>500 IU/L and blood phosphorus <1. 5 mmol/L were selected as MBD group and premature infants with birth weight <1500 g were selected randomly as non-MBD group. General data, pulmonary surfactant, continuous positive airway pressure, mechanical ventilation, start time of enteral nutrition, parenteral nutrition ( PN) time, breast feeding time and breast milk fortifier adding, drug usage, hospitalization time and complications were re-corded and compared between the two groups. Results A total of 440 premature infants with birth weight<1500 g were admitted to the hospital during the study period. 58 [ 13. 2％ ( 58/440) ] infants were enrolled in the MBD group, among which infants with birth weight<1000 g accounting for 56. 9％ ( 33/58) . High birth weight (OR=0. 62, 95％ CI:0. 389-0. 990) was an independent protective factor of MBD in premature in-fants. The higher the birth weight, the lower the risk of MBD in premature infants. The longer duration of breast feeding time ( OR= 2. 191, 95％ CI:1. 628-2. 950) , later initial time of enteral feeding ( OR=2. 695, 95％CI:1. 710-4. 248), longer duration of PN (OR=6. 205, 95％ CI:3. 359-11. 463) time, longer duration of respiratory supporting time ( OR=1. 046, 95％ CI:1. 026-. 067) , longer hospital stay time ( OR=1. 703, 95％ CI:1. 109-2. 615) and small for gestational age ( OR=2. 965, 95％ CI:1. 163-5. 658) were inde-pendent risk factors of MBD in premature infants. The duration of PN was the most important independent risk factor of MBD in premature infants (OR=6.205, 95％ CI: 3.359-11.463). Conclusion Multiple factors can lead to MBD of premature infants. The high birth weight is an independent protective factor of MBD and the duration of PN is the most important independent risk factor of MBD in premature infants.

Objective:To study the effects of different sizes of maternal placental chorionic hemangioma (PCH) on neonatal clinical outcome.Methods:February 2013 to December 2020, neonates whose mothers with PCH delivered in our hospital were retrospectively analyzed. According to the diameter of PCH, the neonates were assigned into giant PCH group (diameter≥4 cm) and ordinary PCH group (diameter<4 cm). Clinical characteristics and outcomes were compared between the two groups.Results:A total of 35 cases were enrolled in the study. 13 cases (37.1%) were male, 12 cases (34.3%) were Cesarean section delivered, 11 cases (31.4%) were premature infants, 12 cases (34.3%) had low birth weight and 12 cases (34.3%) were admitted to NICU, 7 cases (20.0%) had intrauterine distress, cardiac enlargement and abnormal hematological indexes, respectively, 6 cases (17.1%) needed respiratory support; 5 cases (14.2%) had increased amniotic fluid and fetal edema, respectively, 4 cases (11.4%) received blood transfusion, 3 cases (8.5%) had postnatal asphyxia, 2 cases (5.7%) had brain injury and 2 cases (5.7%) had congenital malformation. 15 cases were in the giant PCH group and 20 cases in the ordinary PCH group. Compared with the ordinary PCH group, the giant PCH group had significantly higher incidences of prematurity, low birth weight, increased amniotic fluid, intrauterine distress, NICU hospitalization, fetal edema, cardiac enlargement, respiratory support, abnormal hematological indexes, blood transfusion and mortality ( P<0.05). Conclusions:Maternal complications with giant PCH may significantly increase the risk of neonatal complications, thus perinatal monitoring should be strengthened.【 Key words】Placental chorionic hemangioma; Infant, newborn; Clinical outcome

Objective:To investigate the effects of nutritional intake in the first two weeks of life on bronchopulmonary dysplasia (BPD) in preterm infants with gestational age (GA) ≤ 32 weeks.Methods:A retrospective case-control study was conducted 154 preterm infants with birth weight ≤ 1500 g and GA ≤ 32 weeks were enrolled from neonatal intensive care unit (NICU) of Affiliated Hospital of Qingdao University between January 1, 2016 and December 31, 2017. These infants were divided into BPD group or non-BPD group. All clinical and nutritional data were collected and analyzed to investigate the effects of early-life (within 2 weeks after birth) nutritional intake on BPD.Results:Among a total of 154 eligible neonates, 68 were without BPD and 86 with BPD (55.8%). Mild, moderate and severe BPD accounted for 39.5% (34/86), 58.1%(50/86)and 2.4%(2/86)of all BPD cases respectively. GA and birth-weight of BPD group were significantly lower than that of non-BPD group [(28.35 ± 1.55)weeks vs. (30.12 ± 1.23)weeks; (1050.91 ± 190.6)g vs. (1205.88 ± 195.83)g, both P = 0.000]. The duration of mechanical ventilation in BPD group was longer than that in non-BPD group [(2.65 ± 1.08)days vs. (0.47 ± 0.12)days, P < 0.05]. The incidences of complications in BPD group, including neonatal asphyxia, sepsis and patent ductus arteriosus, were all higher than those in non-BPD group( P < 0.05). The fluids and caloric intake, enteral fluids and caloric intake were significantly lower in BPD group on Day 7 and 14 of life ( P < 0.05). The macronutrient intake in BPD group was also consistently lower, reaching statistical significance for carbohydrate intake on Day 7 and 14 of life, and for protein and lipid intake on Day 14 of life ( P < 0.05). Multivariate logistic regression analysis showed that mechanical ventilation ( OR = 2.257, 95% CI: 1.143~4.456, P = 0.019) and GA ( OR = 0.325, 95% CI: 0.215~0.49, P = 0.000) were high-risk factors for BPD. The decreased odds of developing BPD were associated with higher levels of enteral calories on Day 14 of life ( OR = 0.96, 95% CI: 0.94~0.98, P = 0.000), fluids on Day 7 of life ( OR = 0.927, 95% CI: 0.876~0.981, P = 0.009) and protein intake on Day 14 of life ( OR = 0.044, 95% CI: 0.011~0.177, P = 0.000). Conclusions:GA and mechanical ventilation were independent high-risk factors for BPD. Higher intake of protein and enteral calories were protective factors. Proactive early enteral nutrition support, adequate protein intake and decreasing the duration of mechanical ventilation may reduce the risk of BPD.

Objective:To study the risk factors and clinical outcomes of early pulmonary hypertension in preterm infants with gestational age(GA)≤32 w.Methods:From October 2017 to May 2021,preterm infants with GA≤ 32 w admitted to NICU of our hospital were retrospectively studied. According to their echocardiography 2 w after birth, the infants were assigned into early-onset pulmonary hypertension (ePH) group and non-PH group. SPSS 21.0 statistical software was used to analyze the general status, complications and clinical outcomes of the two groups. Multiple logistic regression was used to analyze the risk factors of early-onset PH.Results:A total of 183 cases were enrolled, including 24 in the ePH group and 159 in the non-PH group. The incidences of birth asphyxia, hemodynamically significant patent ductus arteriosus (hsPDA), FiO 2≥30% within 6 h after birth, late-onset PH, severe bronchopulmonary dysplasia(BPD) and intracranial hemorrhage(ICH) in the ePH group were significantly higher than the non-PH group( P<0.05). hsPDA was the independent risk factor for early-onset PH ( OR=11.781, 95% CI 4.192-33.108). Conclusions:Preterm infants with GA≤32 w and early-onset PH are at increased risks of ICH, late-onset PH and severe BPD, hsPDA is the independent risk factor for early-onset PH.