Objective To develop a sensitive and stable substrate for enzyme‐linked immunosorbent assay(ELISA)and carry out methodology evaluation tests on it .Methods A kind of substrate with tetramethyl benzidine and urea peroxide as its main compo‐nents were made up ,its sensitivity ,precision and storage stability were evaluated and compared with other three kinds of substrate . Results This substrate was more sensitive than two of the three commercial substrates .The variable coefficient of precision test was 3 .5% .The optical densities(OD) of the new substrate after stored one day ,seven days ,one month ,six months ,twelve months in 4 ℃ environment were 2 .268 、2 .403 、2 .358 、2 .278 、2 .330 respectively ,the standard deviation was 0 .056 185 ,the variable coeffi‐cient was 2 .4% .Conclusion The substrate proposed in this paper displays good sensitivity ,precision and stability .The preparation method is simple ,reproducible .This substrate not only satisfies the requirements of laboratory research ,but also meets with the de‐mands for commercial kit development due to its function as an important component of test kits .It is an economical and effective choice for both laboratories and research transformation .

Objective To explore the mechanism and therapeutic effects of wet dressing with 50％ magnesium sulfate ( MgSO4 ) as a part of comprehensive treatment for wound resulted from pit viper bites in children.Methods A total of 61 children with pit-viper-bitten wound enrolled from May 2009 through May 2011 were divided into two groups,namely the comprehensive treatment group (n =31 cases) and the conventional treatment group (n =30 eases). In the comprehensive treatment group,50％ magnesium sulfate wet dressing applied to the swelling parts of body in the early stage in addition to the conventional treatment and topically bandaged once a day keeping wet with spraying saline on the top of dressing.Before the treatment and on the 1st day,the 3rd day and the 5th day of treatment,the values of myocardial enzyme and the circumference of swelling parts were recorded,and the differences were analyzed by t- test.The differences in rate of wound healing in one week between two groups were analyzed by chi-square test.Results Before the treatment,there was no statistical difference in clinical features between the two groups with different modes of treatment for the viper-bitten children (P ＞ 0.05 ). During comprehensive treatment,the values of myocardial enzyme and the circumference of swelling parts decreased more markedly than those in conventional treatment group ( P ＜ 0.05 ).The rates of wound healing in one week of two groups were 80.5％ and 56.6％ respectively.Conclusions The comprehensive treatment with adjuvant of 50％ MgSO4 for the viper-bitten children can effectively reduce the tissue damage caused by venom,and the swelling as well,restoring function of body and shortening the time for wound healing and preventing complications.

Objective:Study the effect of Acanthopanax gracilistylus extract(Age) on monocyte,and reveal the anti trmor mechanism.Methods:The content of TNF ?,IL 12 were examined by using biological method and ELISA,respectively.The expression of monocytic cytokine mRNA are investigated by RT PCR.The effect of Age on the phagocytosis of monocytes is studied using TdR incorporation method.Results:After cultured with Age,the production of cytokines by monocytes such as TNF ?,IL 12 are markedly increased with good dose dependent manner.After monocytes cultured with LPS in the presence or absence of Age,Age markedly enhances LPS induced TNF ? production.Treatment with Age,the production of cytokines and the expression of their mRNA are increased simultaneously.The results in vivo also show:Age can enhance the productions and the expressions of TNF ? and IL 12.Conclusion:The anti tumor active component of Age has obvious stimulative activity on monocytes, increasing the production of cytokines, enhancing the phagocytosis.These results product the scientific evidence for the study of anti tumor mechanism and clinical ultilization.

Objective To investigate pulmonary function in terms of tidal respiration and the influencing factors on it in ＜ 34 weeks premature infants with RDS at corrected gestational age of 40 weeks.Methods A total of 49 of ＜ 34 weeks premature infants with RDS (RDS group) and 36 of ＜ 34 weeks premature infants without RDS (non-RDS group) followed throughout entire twelve months were collected fromn December 2013 to October 2015.Of them,35 RDS patients and 20 non-RDS patients had the pulmonary function examination.A total of 26 full term infants with hyperbilirubinemia (full term group) were recruited for comparison study.The differences in parameters of tidal respiration were compared among the three groups.The RDS patients were further divided into the mild RDS subgroup and severe RDS subgroup according to the severity of illness.Result ①The TPEF [(0.17 ± 0.04) s vs.(0.23 ± O.09) s],VPEF [(6.74±2.70) mLvs.(9.33±2.92) mL],TPEF/TE [(29.06±4.21)％ vs.(38.27± 7.16) ％],VPEF/VE [(32.54 ± 4.43) ％ vs.(39.64 ± 5.88) ％] in RDS group were significantly lower than those in full term group (P ＜0.05).The TPEF [(0.19 ±0.06) s vs.(0.23 ±0.09) s],TPEF/TE [(30.31 ±11.53)％ vs.(38.27±7.16)％],VPEF/VE [(34.39±8.44)％ vs.(39.64±5.88)％] in non-RDS group were significantly lower than those in full termn group (P ＜ 0.05).The TPEF,TPEF/TE,VPEF/VE in RDS group were lower than those in non-RDS group,but the differences were not significant (P ＞ 0.05).②The TPEF,VPEF,TPEF/TE,VPEF/VE in mild RDS group were higher than those in severe RDS group,but the differences were.not significant (P ＞ 0.05).③ Logistic regression analysis indicated that the gestational age was the protective factor of pulmonary function in premature infants with RDS.Conclusions Small airway resistance in ＜ 34 weeks premature infants with RDS is higher than that in full term infants.There was no significant difference in pulmonary function between RDS premature infants and non-RDS premature infants.The gestational age was the influencing factor of pulmonary function in premature infants with RDS.

Moyamoya disease is a rare cerebrovascular disorder, characterised by progressive stenosis and/or occlusion of the intracranial internal carotid artery and its proximal branches with the development of a basal collateral network. Moyamoya disease has a high prevalence in Asia, particularly in Japan, Korea and China. Ischemic events and intracranial bleeding are the most common clinical manifestation of moyamoya disease. Although the benefi cial effect on hemorrhage is still not clear, revascularisation surgery remains the most effective way to prevent the progression of ischemic symptoms. Moyamoya disease has been investigated by numerous studies since it was fi rst described 50 years ago, many conundrums remain to be solved. In this article, we review the history, epidemiology, aetiology, clinical manifestation, diagnosis and treatment of moyamoya disease. Recent advances and future challenges of moyamoya disease are also discussed.

The distinctive LT siRNAs were designed according to the LT sequence .During the process of cultivation ,siRNA targeting the LT gene ,non‐specific control siRNA ,negative control siRNA and culture medium were added into siRNA group (siRNA‐LT1 group , siRNA‐LT2 group) ,siRNA‐coa3 group ,siRNA‐NC group and blank control group ,respectively ,and three times in each group (1 nmol each time) .After siRNA added at the first time ,bacteria was collected in 45 min (A) ,90 min (B) and 135 min (C) time points .The expression of mRNA in three time points (A ,B and C) were detected by real‐time fluorescence quantitative PCR .The protein level of LT in siRNA‐LT1 group ,siRNA‐LT2 group and blank control group were detected by Western blot in three time points .Results The results of real‐time fluorescence quantitative PCR showed that inhibition of siRNA‐LT1 on the expression of LT mRNA at the three time points(A ,B and C)were 70 .9% ,70 .1% ,72 .5% respectively ,and inhibition of siRNA‐LT2 on the ex‐pression of LT mRNA at the three time points(A ,B and C)were 70 .1% ,69 .2% and 70 .5% respectively .In the three time points (A ,B and C)the inhibition rate of the expression of LT mRNA in siRNA‐LT1 group and siRNA‐LT2 group were statistically lower than that in the siRNA‐NC group ,siRNA‐coa3 group and blank control group (P<0 .05) .The results of Western blot showed that in siRNA‐LT1 group the inhibitory rate of expression of LT protein in the three time points were 43 .1% ,18 .4% and 5 .0% ,re‐spectively ;in the siRNA‐LT2 group were 38 .2% ,15 .4% and 30 .1% ,respectively .Conclusion The specific siRNA could inhibit the expression of LT gene in vitro .

Objective To develop a rapid method based on ARMS, named four primers allele-specific PCR(4p-AS PCR), to detect the most common non-deletional ?-thalassemia mutation Hb Constant Spring(Hb CS) by PCR technique. Methods The 4p-ASPCR was used to detect the Hb CS mutation of ?-thalassemia in 38 DNA samples of Hb H disease patients and using PCR-RE and DNA sequencing to confirm the results. Results Among the 38 Hb H disease patients 15 cases was revealed to carry Hb CS, 16 cases were deletional Hb H, and 7 cases need to be defined.Conclusion A simple, rapid and reliable method, named 4P-ASPCR, to detect Hb CS mutation have been developed. It is also may be useful in screen other point mutations such as Hb Quong Sze.

AIM: To clone human ?-globin gene carrying a thalassemic mutation IVS II654(C→T) and establish a eukaryotic expression system for high-level expression of human ? IVS II654 gene in mouse erythroleukaemia(MEL) cells. METHODS: The fragments of human ? 654 gene isolated from the ? thalassemia patients homozygous for the ? 654 mutation were amplified by PCR, and cloned to plasmid pBGT51. Then, the human ? LCR and ? 654 gene were subcloned from plasmid pBGT51 to the stable mammalian expression vector pcDNA3.1+ together, and the MEL cells were transfected with this vector using commercially available cationic lipid FuGENE6. The MEL cells were induced for further maturation by DMSO and the expression of human ? 654 gene in the MEL cells was identified by RT-PCR. RESULTS: A mammalian expression system of human ? thalassemic mutation ?IVS II654(C→T) was established. CONCLUSION: The level and the reliability of expression of human ? 654 gene in the MEL cells in vitro are similar to that in vivo in human body. This may be a valuable gene therapy model for human ? thalassemic mutation ?IVS II654(C→T).

AIM: The purpose of this study was to explore the expression of estrogen receptor(ER) and progesterone receptor(PR) mRNA in endometrium of rats with endometriosis. METHODS: The rat model of endometriosis was established, and the expression of ER, PR mRNA in the endometrium was examined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The expression of ER and PR mRNA in ectopic endometrium was significantly lower than that in eutopic and normal endometrium (P

Objective To explore how the severity of periventricular-intraventricular hemorrhage (PIVH)impact on physique and neurodevelopment in premature infants.Methods A total of 80 preterm infants with PIVH admitted to NICU of Qingdao Women and Children's Hospital from December 2013 to June 2015 were eligible.According to the Papile classification,the premature infants were divided into 4 groups.They were grade Ⅰ PIVH group,grade Ⅱ PIVH group,grade Ⅲ PIVH group and grade Ⅳ PIVH group.The infants with grade Ⅰ PIVH and grade Ⅱ PIVH belong to the low-grade PIVH group.The infants with grade Ⅲ PIVH and grade Ⅳ belong to the severe-grade PIVH group.All of them were regularly followed up for 12 months.Neurodevelopmental outcomes of infants at 6 and 12-month correction age were assessed by using the 20 items neuromotor assessment applying for 0-1 year old and the Bayley scales of infant development-Ⅱ.The differences in physical and neurophysical development of premature infants among 4 groups were compared.Results There were no significant differences in physical growth indicators such as body weight,body length and the incidence of weight growth retardation among 4 groups (all P＞0.05).The incidence of neurobehavioral abnormalities in infants with grade Ⅲ-Ⅳ PIVH was significantly higher than that of infants with grade Ⅰ-Ⅱ PIVH at 12-month correction age (21.05％ vs 3.28％,x2 =4.284,P=0.038).Physical development index(PDI) of grade Ⅰ-Ⅱ PIVH infants was significantly higher than that of grade Ⅲ-Ⅳ PIVH infants at 6-month correction age(F=11.500,P＜0.05).At 12-month correction age,grade Ⅰ-Ⅱ PIVH infants showed a significant higher mental development index(MDI) scores and PDI scores than those of grade Ⅲ-Ⅳ PIVH infants(F=14.227,16.515,all P＜0.05).Of the 80 cases assessed,infants with grade Ⅲ-Ⅳ PIVH had significantly higher rates of cerebral palsy(21.05％ vs 1.64％,x2 =6.300,P=0.012) and developmental delay (26.32％ vs 4.92％,x2=5.185,P=0.023) compared with grade Ⅰ-Ⅱ PIVH infants.Conclusions The severe PIVH can have negative effect on the neurodevelopmental outcomes of preterm infants and might induce mental retardation,cerebral palsy and other neurodevelopmental disabilities.Therefore,the regular follow-up and early intervention in preterm infants with PIVH should be implemented to improve the quality of their lives.