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 investigate pulmonary function of tidal respiration in late premature infants with non-mechanical ventilation treatment at corrected gestational age of full term. Methods A total of 26 late premature infants with non-mechanical ventilation treatment at corrected gestational age of full term were collected from Qingdao Women and Children′s Hospital between April and December 2013,and saved as the late premature infant group. Also, a total of 31 full term infants with hyperbilirubinemia or neonatal ABO hemolytic disease were recruited and treated as the full term group. Then the parameters of pulmonary func-tion of tidal respiration were compared between the two groups. The late premature infant group was equally allocated into extrauterine growth retardation and non-extrauterine growth retardation groups according to the increase in weight and the ratio of time to peak tidal expiratory flow and total expiratory time(TPEF/TE) and the ratio of expiratory volume at peak tidal expiratory flow and total expiratory volume( VPEF/VE) were respectively compared in these two groups. Another correlation analysis between body weight and TPEF/TE was carried out in the non-extrauterine growth retardation group. Results (1)Gestational age difference had no significant significance between late premature infant group and full term group ( P <0. 05 ) . The body weight[(2. 39 ± 0. 44)kg vs. (3. 21 ± 0. 43) kg] and height[(46. 51 ± 3. 22) cm vs. (50. 16 ± 2. 14) cm] in late premature infant group were still lag behind that in full term group and the difference had significant significance(P<0. 05,respectively). (2)The comparison of parameters of pulmonary function of tidal respi-ration:the TPEF/TE [ ( 41. 74 ± 10. 94 )% vs. ( 48. 17 ± 11. 79 )%] , VPEF/VE [ ( 42. 66 ± 9. 66 )% vs. (48. 31 ± 9. 94)%],VPEF[(8. 02 ± 2. 85) ml vs. (10. 23 ± 3. 56) ml] and minute ventilation[(0. 76 ± 0. 23) L/min vs. (1. 00 ± 0. 44) L/min] in late premature infant group were significantly lower than those in full term group(P<0. 05,respectively). (3)The TPEF/TE and VPEF/VE all had significant significance in both late premature infant group and full term group(late premature infant group:r=0. 958,P=0. 000; full term group:r=0. 950,P=0. 000). (4)The TPEF/TE and VPEF/VE in non-extrauterine growth retardation group were closer to those in full term group. The TPEF/TE and VPEF/VE in extrauterine growth retardation group were lower than those in non-extrauterine growth retardation group(t= -2. 775,-2. 109,P=0. 011, 0. 047). Conclusion Airway(especially small airway) resistance of late preterm infant group is higher than that of term infants. Extrauterine growth retardation influences the airway(especially small airway) resist-ance.

To discuss bacteria of urine infection reoccurrence , the routine culture and L-form bacteria culture of urine tract infection on 850 cases were adopted and clinica l watching and analysis of the bacteria plasmid were held ．Result: of the l28 cases, re-infec tion rate l5.0%．,the positive rate of the routine bacteria culture for those initial diagnosis patie nts was 58.3% while that of the re-infection patients was 34.0% P<0.0l ．The initia l diagnosis L-form inspected rate 5.6%yet that of re-infection was up to 32.0 % P<0.01 ．The analysis of the pla smid bacteria of re- infection were of the same origins ．Conclusion: the first infection dont nee d L-form culture but re- infection should have L-form culture ．Analysis: plasmid positive bacteria of r e-infection was the result of the first infection not completely cured．

Objective To establish a simple,accurate and practi cal method of extracting plasmid from gram-negative and gram-positive bacteria ,Method we selected,Biswajit sahas method and comported others method, such as controlling pH andtemperature strictly and increasing EDTA-Na2 capacity． Result The eight strip s of Ecliv 5l7 can not be found completely in original method, but can be found in our one．We found the largest plasmid molecular weight was l86.3?kb, and t he minimum was l.l?kb from each 30 strains STA, EC, PA． Plasmid profile is alike through repeating ex p eriment three times． Conclusion:The method can be widely used in extracting bac teria plasmid and fit gram-negative and gram-positive bacteria whenever molecular weight ma x or min． These conditions can be obtained in general laboratory．

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.

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 .

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

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.