OBJECTIVE:Using molecular techniques, we typed 2 short tandem repeat (STR) loci on 21 chromosome to establish the method for rapid and accurate diagnosis of trisomy 21.METHODS:Genomic DNA samples from 50 individuals diagnosed previously by karyotype as trisomy 21 and 40 children with severe mental retardation (IQ < 50) suspected of trisomy 21 were analyzed for 2 short tandem repeat loci on 21 chromosome, D21S1435 and D21S2055. Typing was carried out after polymerase chain reaction (PCR) and silver staining. The trisomy was identified by the number of alleles: 3 alleles bands whose density is same, two alleles bands with one obvious higher density compared to the other and one allele band whose density is three times than the normal control.RESULTS:All of the complete trisomy 21 were detected by this method; the parental source was easily determined.CONCLUSION:This method for diagnosing trisomy 21 is rapid and accurate.
OBJECTIVE:To construct a recombinant BCG secretively expressing ESAT-6 of Mycobacterium tuberculosis.METHODS:alpha-antigen(alpha-Ag) signal sequence and esat-6 gene were amplified from the genome of Bacille Calmette-Guerin (BCG) and Mycobacterium tuberculosis by PCR respectively. esat-6 gene was cloned in E. coli-BCG shuttle-plasmid pMV261 to get pME. Then a new recombinant plasmid pSME was constructed by inserting BCG alpha-Ag signal sequence into pME.RESULTS:The cloned genes alpha-Ag signal sequence and esat-6 were correctly inserted into the vector pMV261, which was confirmed by restriction endonuclease digestion and PCR amplification of pSME.CONCLUSION:pSME was expected to secretively express ESAT-6 of Mycobacterium tuberculosis in BCG. This study provides the possibility of further researches on the development of new anti-tuberculosis vaccine.
OBJECTIVE:To investigate the structure and function of human nerve growth factor (beta-NGF) and the gene encoding beta-NGF.METHODS:A pair of specific primers (29 mer) for the sequence encoding human beta-NGF was designed and synthesized. A 380 bp fragment was amplified from human blood genomic DNA by polymerase chain reaction, and cloned into pGEM-T Easy vector. The identified insert fragment from the recombinant pGEM-T-NGF was directionally ligated with linearized pGEX-5T with the compatible termini. E. coli JM 109 was transformed with the expression recombinant p5TNGF and induced by IPTG.RESULTS:The cloned DNA fragment was identified as the full-length sequence encoding human beta-NGF by restriction analysis and DNA sequencing. SDS-PAGE and Western blot revealed the cloned NGF gene expressed as a fusion protein (40.5 x 10(3) u) in the cells transformed by p5TNGF. The soluble fusion protein was determined to be 503.2 mg/L, accounting for 6.8% of the total soluble protein (7.4 g/L) of bacterial cells. This fusion protein was found to have antigenic activities of NGF.CONCLUSION:The clone containing the full-length sequence encoding human beta-NGF is obtained and successfully expressed in E. coli to be of use for studying the biological functions of human beta-NGF gene.
OBJECTIVE:To investigate the serum lipid and apolipoprotein levels in pregnancy-induced hypertension (PIH or pre-eclampsia), normotensive pregnant women and non-pregnant women.METHODS:Twenty-eight women with PIH, 31 normatensive and 24 non-pregnant ones were studied, from whom maternal blood samples were collected. Serum lipid levels were measured by enzymatic method and apolipoproteins AI, B100, C II, CIII and E levels by radial immunodiffusion kits developed by our Lab.RESULTS:1. Serum TG, apoC II and apoC III were highly increased in normotensive pregnant women by 3.5, 2.4 and 2.8 times respectively, when compared with those in non-pregnant ones (P < 0.001); serum TC, nHDL-C and apoAI, B100 and E were also increased in normotensive pregnant women by 21%, 33%, 55%, 79% and 77% respectively, when compared with those in non-pregnant ones (P < 0.001). TG/HDL-C in normotensive pregnant women was also significantly, higher than that in non-pregnant ones(P < 0.01). Serum HDL-C levels remained unchanged in both groups. 2. Serum TG, apoC II and apoC III were highly increased in PIH patients by 2.3, 4.0 and 2.8 times respectively, when compared with those in non-pregnant ones (P < 0.001); serum TC, nHDL-C and apoAI, B100 and E were also increased in PIH patients by 27%, 26%, 52%, 90% and 67% respectively, when compared with those in non-pregnant ones (P < 0.001) TG/HDL-C in PIH patients was also significantly higher than that in non-pregnant ones (P < 0.01). Serum HDL-C levels were increased in PIH patients by 29%, when compared with non-pregnant women. Only serum HDL-C level in PIH patients was higher than that in normotensive pregnant women. No significant differences were seen in other lipid and apolipoprotein levels between PIH patients and normotensive pregnant ones. 3. There was no significant difference in the levels of serum lipid and apolipoprotein between the subgroups of PIH patients according to the severity of the disease.CONCLUSION:The increase of serum lipid and apolipoprotein levels in PIH is probably not the predictor for pre-eclampsia.
OBJECTIVE:To establish a specific method for determination of IgG in IVIG.METHODS:The IVIG samples were disposed by acylation and analyzed by Rocket Immunoelectrophoresis. The amount of the IgG in IVIG was calculated by an IgG standard curve.RESULTS:The standard curve by this method has a very good linear relationship: r = 0.9967. The reproducibilities with 3 bathes of IVIG samples (n = 6) are 5.37% +/- 0.21%, 5.28% +/- 0.39% and 5.80% +/- 0.30%.CONCLUSION:This method could be adapted to regular assay in blood products.
OBJECTIVE:To evaluate the efficacy and safety of domestic sparfloxacin.METHODS:A multicentre randomized controlled clinical trial was conducted to compare sparfloxacin versus ofloxacin in the treatment of acute bacterial infections. 58 patients received 200 mg of sparfloxacin once daily intravenous drip and 61 patients received 200 mg of ofloxacin twice daily intravenous drip. Both drugs were given for 7-14 days.RESULTS:The clinical cure rates of sparfloxacin group and ofloxacin group were 44.83% and 42.62%, and the efficacy rates were 87.93% and 81.97%, respectively; no significant differences were noted between the two groups (P > 0.05). The bacterial eradication rates were 86.00% and 84.62% respectively (P > 0.05). The adverse reactions in the two groups were 13.79% and 22.58% (P > 0.05). Photosensitivity reaction was not observed in this study.CONCLUSION:Domestic sparfloxacin is effective and safe in treating acute bacterial infections.
OBJECTIVE In order to search for more effective treatment regimen of chronic myeloid leukemia (CML). METHODS Semisolid culture and conventional chromosome assay were used to evaluate the effect of interferon (IFN) and Ara-c on K562 cell line and BMMNCs from CML patients and normal controls. RESULTS IFN had no obvious inhibitory effect on K562 cell. IFN and Ara-c had preferential inhibitory effect on CFU-GM growth of CML and selective cytotoxic effect on CML Ph+ cell. The combination of IFN with Ara-c enhanced the cytotoxic effect of IFN or Ara-c alone on CML cells. CONCLUSION IFN and Ara-c can be selected for therapy of CML in chronic phase. The combination of IFN with Ara-c may be more effective than IFN or Ara-c alone for the therapy of CML.
OBJECTIVE:To study the effect of a calcium channel antagonist--Nifedipine(NIF) on the prevention and treatment of premature labor, the suppression of uterine contraction, the prolongation of pregnancy, and to observe the side effects of NIF.METHODS:Eighty-four patients with threatened premature labor were randomly divided into three groups according to the dosage of NIF. Group A received NIF according to a 10 mg dosing schedule; Group B received NIF according to a 20 mg dosing schedule; Group C(not treated with NiF) served as control. The time of delivery delayed, the relationship of therapeutic effect with cervical dilatation and gestational age, and the results of delivery were observed and analyzed.RESULTS:In this study, INF effectively suppressed uterine contraction and delayed the time of delivery. The suppressant intensity was associated with the dosage. The therapeutic effects was apparently more noticeable when the treatment was initiated before the active opening of cervical os. No side effect of NIF was observed in Groups A and B.CONCLUSION:Nifedipine is highly effective as a tocolytic agent for the management of premature delivery. It is of clinical usefulness, and the results of the above observations merit further study.
OBJECTIVE:To aim at demonstrating whether cationic liposome-mediated antisense c-myb oligonucleotide(LipoAON) can inhibit the growth of C6 glioma by intravenous injection.METHODS:Intracerebral C6 glioma cells were implanted into the left caudal nucleus of forty-eight male Wistar rats. There were four groups: LipoAON(n = 12), antisense c-myb oligonucleotide (AON; n = 12), cationic liposome (Lipo; n = 12), and normal saline (NS; n = 12). Six days after tumor implantation, the above-mentioned drugs were injected into the right femoral veins of the rats respectively. Two days later, the same drugs were injected into the left femoral veins. The appetite, motor and weight of every animal were closely observed during the whole experiment. Six rats of each group were respectively killed 4 days and 10 days after the end of administration. The weight change, pathologic examination and immunohistochemical analysis of c-myb expression of the tumor were completed.RESULTS:In LipoAON group, the growth of the tumors was significantly inhibited in a short time after treatment and c-myb expression was down-regulated. But in the AON group and Lipo group, the growth of the tumors was not inhibited and c-myb expression was not down-regulated, compared with that in NS group. The inhibitory effect of LipoAON on the tumors rapidly declined with time and c-myb expression was again up-regulated.CONCLUSION:1. Cationic liposome (LipofectAMINE) as transfection vehicle makes c-myb easily penetrate BBTB and enter the tumor. The technique is simple, safe, highly effective for the transfection of c-mybAON; 2. LipoAON has marked inhibitory effect on the growth of C6 glioma. The AON technical method for inhibiting the expression of c-myb oncogene has a research perspective in the treatment of glioma; 3. The inhibitory effect of LipoAON on the growth of glioma declines with time. The question about how to make c-myb AON have highly effective, sustained and stable expression in the tumor still requires further research.
OBJECTIVE:To explore the relation between insulin resistance(IR) and A1166C molecular variant of type 1 angiotensin II receptor(AT1R) gene in Chinese subjects with coronary heart disease(CHD).METHODS:IR was calculated by 1/(fast plasma insulin x fast plasma glucose). The A1166C molecular variant of AT1R gene was determined by polymerase chain reaction and restriction endonuclease analysis methods in 90 patients with coronary heart disease and 80 healthy people.RESULTS:The frequency of allele C of group CHD was significantly higher than that of control group. There was no significant difference of IR level among AT1R A1166C AA, AC, CC genotypes in patients with CHD(P > 0.05).CONCLUSION:No significant association between A1166C molecular variant of AT1R gene and insulin resistance was found in Chinese with CHD.
OBJECTIVE:To investigate the expression and mutation of MTA1, nm23H1 and E-cadherin(E-cad) genes in ovarian carcinoma (OC) in relation to lymph node (LN) metastasis.METHODS:A panel of normal ovarian tissues, primary OC specimens and corresponding LNS was examined for mRNA expression and mutation of MTA1 and nm23H1 and protin expression of E-cad genes by using RT-PCR, RT-PCR-SSCP and immunohistochemistry.RESULTS:The frequency of MTA1 over expression was 100%(7/7) in primary OC with metastasis but only 38.5%(5/13) in those without metastasis (P = 0.0103). Overexpression of MTA1 was observed in 87.5%(6/7) of LNS with metastasis but in only 23%(3/13) of LNS without metastasis (P = 0.0118). In contrast with MTA1, low expression of nm23H1 mRNA was seen in 7 of 7 OC with metastasis but only in 4 of 13(30%) of those without metastasis (P = 0.0043). Low nm23H1 expression was also seen in 7 of 7 LNS with metastasis but only in 5 of 13 (38.5%) nonmetastatic LNS (P = 0.0102). Meantime, no expression of E-cad protein was observed in 7 of 7 OC with metastasis but in 6 of 13(46.2%) of those without metastasis (P = 0.044). In correlation analysis of the three genes, MTA1 reversely correlated with nm23H1 and E-cad respectively (r = -0.903, -0.803), and positive correlation existed between nm23H1 and E-cad (r = 0.724). No mutation of MTA1, nm23H1 and was found by SSCP analysis.CONCLUSION:The mRNA expression of MTA1, nm23H1 and E-cad is positively and negatively correlated with LN metastasis. The expression abnormalities but not the mutations of the three genes are frequent events related to LN metastasis of ovarian cancer.
OBJECTIVE:To study the expression of glucocorticoid receptor (GR) isoform in the peripheral blood mononuclear cell (PBMC) of patients with Graves' ophthalmopathy (GO). METHODS:Semi-quantitative RT-PCR was used to analyze RNA isolated from PBMC of the patients with GO and the normal volunteers. The level of plasma total cortisol (PTC) at 8 a.m. was measured. RESULTS:The hGR alpha/GR beta mRNA ratios of the two groups were 7.58 +/- 5.42 and 14.65 +/- 8.30, respectively. There was significant difference between the two groups (P < 0.05). The PTC levels of the two groups were 304.23 +/- 92.06 and 313.73 +/- 111.05, and no significant difference between them was noted (P > 0.05). No correlation was found between hGR alpha/GR beta mRNA and PTC levels. CONCLUSION:hGR alpha/GR beta mRNA may play a role in the pathogeny of Graves' ophthalmopathy.
OBJECTIVETo make a comparision of cell function between the mesangial cells from rats with diabetic mellitus (DMC) and the mesangial cells stimulated by high glucose (HMC) and to explore the feasibility and advantage of culturing mesangial cells (MC) from diabetic rats in studies on the pathogenesis of diabetic nephropathy.METHODSDMC were obtained from rats with streptozotocin-induced diabetes and were cultivated in normal medium. The MC from normal rats were divided into HMC high glucose and NMC cultured in normal medium as control. The cellular proliferation was assessed using 3H-thymidine incorporation. Production of fibronectin (FN) was analysed by ELISA. And with the use of laser scanning confocal microscopy, the calcium concentration ([Ca2+]i) and the response of [Ca2+]i to Angiotensin II (Ang II) were measured.RESULTSThe 3H-TdR incorporation efficiency of DMC was significantly higher than that of CMC; the 3H-TdR incorporation efficiency of HMC was lower than that of CMC (P < 0.05). The secretion of FN in DMC and HMC was higher than that in CMC (P < 0.01 and P < 0.05 respectively). The response of [Ca2+]i to Ang II in DMC and HMC was decreased (P < 0.05), and the decrease was more significant in DMC than in HMC.CONCLUSIONSThe function and biologic character of mesangial cells cultured from diabetic rats are more similar to those of mesangial cells from diabetic in vivo. The result suggest that establishing a diabetes model first and starting the culture of MC next is probably a good method for investigating the mesangial cells in diabetic nephropathy.
OBJECTIVE:To explore the mechanism of ischemia reperfusion injury (IRI) and the protective effect of Yisheng injection on endothelial cells (ECs).METHODS:Human umbilical veins endothelial cell line ECV304 was cultured in RPMI medium 1640 without glucose under 100% N2 for one and a half hours, and then in RPMI medium 1640 with glucose under normal conditions for 5 hours to mimic ECs' IRI posttransplantaion. A pure nature medicine, Yisheng injection was added into the medium in experimental group. Intracellular calcium and mitochondrial were shown through flourescent staining. The activities of succinic dehydrogenase (SDH) and lactic dehydrogenase (LDH) were detected by cytochemical staining. The concentrations of superoxide dismutase (SOD), malonaldehyde (MDA) and nitrous oxide (NO) in culture medium were determined.RESULTS:After treatment with anoxia-reoxygenation, the ECs' morphologic changes were observed, and intracellular calcium concentration, LDH activity, MDA concentration became higher. Meanwhile, mitochondrial membrane potential, concentrations of SOD and NO were lower. Yisheng injection made these changes disappear or become less.CONCLUSION:Anoxia-reoxygenation induces lipid peroxidation, calcium superrcharge in ECs, and damage to mitochondrial function. Yisheng injection can reverse these changes, thus protecting the endothelial cells.
OBJECTIVE:To investigate the effect of Tanshinone II A on K562 cell-line and its possible mechanism.METHODS:Cell culture, cytomorphology and cytometrics were used.RESULTS:The proliferative inhibition of K562 cells induced by Tanshinone II A was dose-independent, and the degrees of erythroid differentiation induced by 30 nmol/L ACM and 0.5 microgram/ml Tanshinone II A were not statistically different (P > 0.05).CONCLUSION:Tanshinone II A has an inhibitive effect on the proliferation of K562 cells and an inductive effect on the differentiation of erythrocyte series. The mechanisms may be related to the changes of cell cycle and gene expressions.
OBJECTIVE To investigate the 192 Gln-Arg polymorphism of paraoxonase (PON) gene and its relationship with serum lipids and apolipoproteins (apo) levels in patients with endogenous hypertriglyceridemia in Chinese population in Chengdu area. METHODS The genotype and allele frequencies of paraoxonase gene 192 Gln-Arg polymorphism were assayed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Serum lipids were measured by enzymatic kits and apolipoproteins AI, A II, B100, C I, C II and E were measured by the RID kits developed by the Apolipoprotein Research Unit of this university in 128 HTG patients whose fasting serum TG levels were > or = 2.26 mmol/L and 129 healthy subjects whose fasting serum TG levels were < 1.82 mmol/L and TC levels < 6.2 mmol/L from a population of Chinese Han nationality in Chengdu area. RESULTS Both in HTG group and control group, the QR genotype of PON gene was the major one, and the frequencies were 0.515 and 0.581 respectively. No differences were found in PON gene Gln-Arg polymorphism between the HTG group and the control group. In the control group, the QQ genotype of PON gene was found to have higher serum apoA I levels, compared with the RR genotype (P < 0.05). But in the HTG group, when compared with the RR genotype, the QQ genotype was found to have lower serum apoA I and A II levels and higher serum apoE levels. CONCLUSION These may be an association of the QQ genotype of the paraoxonase 192 Gln-Arg polymorphism with the decrease of serum apoA I level and the increase of serum apoE level in endogenous hypertriglyceridemica.
OBJECTIVE:To investigate the expression of insulin receptor in human hepatocellular carcinoma and it's adjacent tissue. METHODS:The human hepatocellular carcinoma and it's adjacent tissue specimens were obtained from 12 patients with histologically confirmed hepatocellular carcinoma at surgery and were immediately frozen under -80 degrees C. Insulin was radioiodinated using Ch-T method. Cell membrane fraction of human hepatocellular carcinoma and it's adjacent tissue were isolated by sucrose density gradient centrifugation. Receptor binding of 125I-insulin to human hepatocellular carcinoma and it's adjacent tissue were performed. Binding data were calculated according to Scatchard using the ligand program. Statistical comparison was made with the paired t-test. RESULTS:The Kd values of human hepatocellular carcinoma and it's adjacent tissue were 2.12 +/- 0.62 nmol/L and 2.21 +/- 0.78 nmol/L respectively; the values of Bmax were 1.94 +/- 0.64 pmol/mg protein and 1.42 +/- 0.57 pmol/mg protein respectively. The Bmax value of human hepatocellular carcinoma was significantly higher than that of the adjacent tissue (P < 0.05), whereas the two Kd values had little difference (P > 0.05). CONCLUSION:The results of this study showed that human hepatocellular carcinoma expressed denser insulin receptors than it's adjacent tissue, but there was no significant increase in the affinity of the carcinoma to insulin.
UNLABELLED:To investigate the possible significance and usefulness of reseeding the endothelial cell of recipient for the prevention of hyperacute rejection (HAR) resulting from the xenograft.METHODS:Discordant xenotransplantation model (guinea pig-to-rat) was adopted in this study. Firstly endothelial cells from rat abdominal aorta were separated and cultured. Secondly the guinea pig abdominal aorta of which the endothelium had been removed was cultured with the suspension containing rat endothelial cells (4 x 10(6)/ml). The viability of cultured vessel was assessed using light microscopy and transmission electron microscopy. Thirdly, the guinea pig vessels reseeded with rat endothelial cells were transplanted to rat in vivo. The thrombrosis time was observed. The condition of deposit of IgM and C3 in the recipient's transplanted vessel endothelium was examined by immuno-fluorescence staining assay.RESULTS:The thrombrosis time of the guinea pig vessel reseeded with rat endothelial cells (20.3 + 4.42 h) was significantly prolonged as compared with that of the normal guinea pig vessel (0.35 + 0.284 h) and the guinea pig vessel that had been deprived of endothelium (0.165 + 0.77 h). No deposit of IgM and C3 was seen in the new endothelium of guinea pig vessel in the treated group, whereas deposit of IgM and C3 was observed in the untreated normal guinea pig aorta.CONCLUSION:Donor vessels reseeded with endothelial cells from the recipient will undergo less severe rejection and this technique may be very useful in the attenuation of HAR.
OBJECTIVE:To improve the primary cell culture methods of colorectal adenomas.METHODS:The methods of the in vitro primary cell culture of colorectal adenomas, mainly the method for taking and preparing specimen, the methods of culture conditions and cells purification, were considered again and improved in this study.RESULTS:Of the 32 cases, of polyp, 26 were successfully cultured and 6 failed. The culture lasted 5-23 days with 50%-95% viscosity of wells. By means of immuno-histochemical staining and electromicroscopy, the cultured cells were proved to be of epithelial origin.CONCLUSION:The primary culture methods of adenoma cells have been improved, but further study on the serial transfer culture cell lines is most needed.
