2000年 · 第113卷第10期

Purpose To review the major achievements of biological products of China in the scientific research and development. Data sources The contents of this article were selected from the original essays or reviews related to biologicals published in Chinese and foreign journals. Study selections Briefly original articles and reviews of text books published by Chinese authors in the last twenty yars were selected. Date extraction These data have addressed the novel vaccines and engineering genetic technologies on biologicals. Results Nowadays about 200 kinds of biological products are produced in China ranging from preventive vaccines as the majority to therapeutic and diagnostic products. About 800-900 million doses of vaccines for the purpose of prophylaxis are administered annually. Biologicals have played an important role in the prevention of infectious diseases. During the former 30 years the basis for the further development of research work was set up and in the latter 20 years new achievements were made one after another, for example, the development of attenuated JE vaccine, hepatitis A live vaccine, HFRS inactivated vaccine, recombinant HB vaccine and Vi typhoid vaccine, et al. Conclusion During the past 50 years, China has made a great progress in the field of biological products, however we are still week in the aspect of basis research and backward behind the developed countries in originating new products. Chin Med J 2000; 113(10): 942-947

Purpose To identify the structure and the function of NMDA receptors, to understand the modulatory mechanism of some endogenous and exogenous compounds on NMDA receptors, and to provide theoretical basis for developing new drugs that modulate NMDA receptors. Data sources A total of 24 originally identified articles were selected. Study selection A total of 24 articles were selected from several hundred original articles or reviews. The content of selected articles are in accordance with our purpose and the authors are authorized scientists in the study on NMDA receptors. Data extraction After careful review of the selected papers, the meaningful results and conclusions were extracted using scientific criteria and our experience in the research of NMDA receptors. Results NMDA receptor contains at least five subunits. They were designated as the NR1(ζ1), NR2A(ε1), NR2B(ε2), NR2C(ε3), and NR2D(ε4). A unique feature of NMDA receptor is the requirement for both glutamate and the co-agnist glycine for the efficient gating. NMDA receptor is modulated by a number of endogenous and exogenous compounds. Mg2+ not only blocks the NMDA channel in a voltage-dependent manner but also potentiates NMDA-induced responses at positive membrane potentials. Na+, K+ and Ca2+ not only pass through the NMDA receptor channel but also modulate the activity of NMDA receptors. Zn2+ blocks the NMDA current in a noncompetitive and a voltage-independent manner. It has been demonstrated that polyamines do not directly activate NMDA receptors, but instead act to potentiate or inhibit glutamate-mediated responses. The activity of NMDA receptors is also strikingly sensitive to the changes in H+ concentration, and partially inhibited by the ambient concentration of H+ under physiological conditions. Conclusions NMDA receptors are glutamate-regulated by ion channels that are permeable to Ca2+, Na+, K+ and are sensitive to voltage-dependent Mg2+ block. This channel complex contributes to excitatory synaptic transmission at sites throughout the brain and the spinal cord, and is modulated by a number of endogenous and exogenous compounds. NMDA receptors play a key role in wide range of physiologic and pathologic processes. Five NMDA receptor subunits have now been characterized in both rat and mouse brain. Chin Med J 2000; 113(10): 948-956

Objective To study the activity of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptors on cAMP production and inward calcium currents(ICa)in guinea pig ventricular myocytes. A comparison was also made with those of a muscarinic receptor agonist. Methods cAMP content was determined by radioimmunoassay and the ICa in guinea pig single ventricular cells were recorded by the whole-cell patch clamp technique. Results Both the muscarinic receptor agonist, carbachol(Carb 10 μmol/L), and anti-peptide antibodies(Abs 100 nmol/L)could decrease basal cAMP levels(by 46.9% ± 4.2% and 60.2% ± 4.6%, respectively)and basal ICa. Both Carb(10 μmol/L)and Abs(100 nmol/L)could also inhibit the isoprenaline-induced(Iso 0.8 μmol/L)increases in cAMP production(from 108.2±7.0 to 88.4±7.2 pmol/mg· protein/min for Carb and 88.6±5.1 pmol/mg· protein/min for Abs, respectively)and the increases in ICa. The muscarinic receptor antagonist atropine(Atr)was able to prevent these effects of Carb and Abs. Conclusions Anti-peptide antibodies against an epitope located in the second extracellular loop of human M2 muscarinic receptors, similar to muscarinic receptor agonist, could decrease the basal ICa and β-receptor agonist stimulated increase of ICa by decreasing the basal and β-receptor agonist stimulated increase of cAMP production, and therefore could have an effect on their target receptor. These results further suggest that autoimmunity may participate in the pathogenesis of human cardiomyopathy and the second extracellular loop of human M2 muscarinic receptor could be the main immunodominant region. Chin Med J 2000; 113(10): 867-871

Objectives To study mitogen-activated protein kinase(MAPKs)activation in fibroblast-like synoviocytes(FLS)of rheumatoid arthritis(RA)under the stimulation of IL-1β, and to elucidate the role of protein tyrosine kinase(PTK)in the activation of MAPKs. Methods Primary cultures of RA FLS were used. Western blot was applied to examine transient changes in protein tyrosine phosphorylation status and MAPKs activation in RA FLS stimulated with IL-1β at various doses, and over different periods. Genistein, the specific PTK inhibitor, was used to evaluate the inhibitory role in activation of MAPKs by IL-1β. Results IL-1β transiently increased protein tyrosine phosphorylation, and activated the MAPKs cascades(mainly ERK2, JNK2 and P38)in RA FLS. There was no obvious difference in MAPKs activation among different doses of IL-1β(1 IU/ml, 10 IU/ml, 100 IU/ml), but the peak activation of ERK2, JNK2 and P38 took place at 5 min, 15 min and 1 min, respectively, after stimulation with IL-1β. The activation of ERK2 was inhibited by genistein, but the inhibitory role on that of JNK and P38 was relatively weak. Conclusions During signal transduction of IL-1β in RA FLS, tyrosine phosphorylation was increased transiently, the MAPKs cascade was activated in a few minutes, and there was heterogenicity in the activation among three subfamily members. PTK had a role in the activation of ERK, but had weak effects on that of JNK and P38. Chin Med J 2000; 113(l0): 872-876

Objective To investigate the imbalance of Th1/Th2-type cytokines in patients with systemic lupus erythematosus(SLE)and its relation to disease activity. Methods Intracellular cytokines were determined by fllow-cytometry following whole-blood culture. Results Patients with systemic lupus erythematosus disease activity index(SLEDAI)> 10 had statistically significantly fewer CD4+ or CD8+ T cells producing IFN-γ than patients with SLEDAI = 0, SLEDAI 1-10 or healthy controls(P< 0.01, P < 0.01 or P < 0.05, respectively). Patients with SLEDAI > 10 also had decreased ratio of IFN-γ/IL-4 positive CD4+ or CD8+ T cells, compared with patients with SLEDAI = 0, SLEDAI 1-10 or healthy controls(P < 0.05). The decreased Th1 or Tc1 cells and the ratios of IFN-γ: IL-4 positive CD4+ T-cells were significantly correlated with disease activity(P< 0.05). Conclusion SLE is characterized by an imbalance of Th1 /Th2 and Tc1/Tc2 cytokines. The decreased Th1 or Tc1 cells and the Th1/Th2 ratio are related to disease activity. Chin Med J 2000; 113(10): 877-880

Objective To clone the full-length of a differentially expressed cDNA fragment, LC27, and study its biological function tentatively. Methods Northern blot was used to analyze the expression pattern of LC27 in hepatocellular carcinoma, matched nontumor liver tissues, fetal liver and normal adult liver tissues, as well as BEL-7402 hepatocellular carcinoma cell line ESTs splicing and 5' rapid amplification of cDNA ends(5' RACE)were used to clone the full-length of LC27 cDNA. An antisense oligodeoxynucleotide approach was used to investigate the biological role of the gene in the proliferation of BEL-7402 cells. Results A 2186 bp novel cDNA with an open reading frame encoding a 283 amino acid protein was cloned. Analysis of the deduced amino acid sequence indicated that it is 38%(88/229)identical to human Golgi 4-transmembrane spanning transporter MTP. The gene and the encoded protein was termed hepatocellular carcinoma overexpressed transmembrane protein(hotp)and HOTP, respectively. Hotp mRNA was almost undetectable in normal adult liver and fetal liver tissues. However, it was significantly up-regulated in hepatocellular carcinoma and some matched nontumor liver tissues, as well as BEL-7402 cells. The proliferation of BEL-7402 cells was suppressed by an antisense oligodeoxynucleotide against hotp mRNA at a concentration of 50 μg/ml. Conclusion HOTP may be an integral membrane transporter protein. The overexpression of the gene in hepatocellular carcinoma may play an important role in hepatocarcinogenesis and disease progression. Chin Med J 2000; 113(10): 881-885

Objective To assess apoptotic status during the development of colorectal cancer and its prognostic value. Methods The apoptotic frequency of 168 fresh adenocarcinoma specimens and primary cultured cells at 2, 12, 24 and 48 hours(9 normal mucosa, 4 adenomas and 9 adenocarcinomas)were measured by flow cytometry(FCM). Apoptotic indices(AI)in situ for 25 adenomas and 77 adenocarcinomas were visualized by TdT-mediated dUTP nick end labeling(TUNEL), Ki-s5 labeling indices(KI), bcl-2, bax, waf1 and p53 were immunostained with ABC method. Results The culture-related apoptosis at 24-48 hours in vitro was obviously decreased in cultured tumor cells when compared with mucosa cells. Spontaneous apoptosis in situ occurred more frequently in tumor with aneuploid type at late stage. There was positive relationship between apoptosis and proliferative activity, determined by both TUNEL and FCM methods. The well-differentiated or early stage lesions with intensive bcl-2/bax expression were significantly more likely to have low AI. p53 accumulation and waf1 depression were mainly related to KI, whereas bax and waf1 overexpression led to a comparatively higher AI/KI ratio. bcl-2 and KI were found to be independent risk factors. Conclusions The data suggest that the depressed susceptibility to inductive apoptosis may contribute to the initial phase of tumorigenesis, and spontaneous apoptosis in vivo may serve as a marker of tumor progression. The bcl-2 and KI may be valuable in predicting prognosis in colorectal cancer. Chin Med J 2000; 113(10): 886-888

Objective To determine whether p16 gene is involved in the genesis of primary hepatocellular carcinoma(HCC). Methods Twenty-five HCC tumor samples with corresponding non-tumor liver tissue specimens were examined for p16 gene alterations. The identification of deletion of exon 1 and exon 2 in p16 gene was performed using comparative multiplex polymerase chain reaction(PCR)analysis. The point mutation of exon 2 in p16 gene was investigated by single strand conformational polymorphism(SSCP)analysis, and the status of p16 gene methylation was screened using a PCR based methylation analysis. 35 parafin-embedded specimens of HCC with corresponding nontumor liver tissues, including the 25 cases described above for screening p16 gene alterations, were investigated for p16 protein expression using immunohistochemical analysis. Results Among 25 cases, 2 homozygous deletions and 1 hemizygous deletion were found in HCC samples. No point mutation was identified in the remaining 22 tumor samples without p16 gene deletions. Hypermethylation was detected in 24%(6/25)of tumor samples. However, the corresponding non-tumor liver tissue specimens were always unmethylated at the p16 locus. Loss of p16 protein expression occurred in 16 of 35(45.7%)tumor samples, and all the non-tumor liver tissue specimens showed positive p16 staining. For the 25 cases examined for p16 gene alterations, the loss of p16 protein expression was observed in all tumors with p16 gene alterations and also in 3 tumors without p16 gene alterations. Conclusion Inactivation of the p16 gene may play an important role in the genesis of primary hepatocellular carcinoma. Chin Med J 2000; 113(10): 889-893

Objective To isolate genes related to blood glucose-control using Sprague Dawley(SD)rat skeletal muscle. Methods Differential gene expression between glucose stimulated and non-glucose stimulated SD rat skeletal muscle was obtained by the differential display(DD)method, Slot blotting hybridization and Northern blot hybridization. Results Several new genes that are differentially expressed in glucose stimulated and non-glucose stimulated SD rat skeletal muscle were isolated. 74 were verified by slot analysis from 181 gene tags isolated. Of them, 33 were cloned and sequenced, and homologous analysis and application for GenBank Access Number were carried out. 21 expressed sequence tag(EST)representing novel genes was confirmed by Northern blot analysis. A total of 9 novel genes showed significant differential expression patterns. Conclusions Using improvements and modifications of the differential display technique, a labor-and cost-saving route was used to identify new genes related to blood glucose-control. We investigated differentially expressed genes at the whole body level instead of the culture cell level, to ensure experimental results closer to the normal physiological state. This technique may be valid in wide-spread application to other related research. Chin Med J 2000; 113(l0): 894-898

Objective To evaluate the impact of kinetics of molecular remission via retro-transcriptase polymerase chain reaction(RT-PCR)assay on the long-term survival in patients with acute promyelocytic leukemia(APL). Methods Seventy patients with newly-diagnosed APL in remission were involved in this study. Monitoring of minimal residual disease(MRD)was performed regularly by RT-PCR assay for PML-RARα during consolidation. Results A 5-year relapse-free survival(RFS)and overall-survival(OS)were estimated as 46.8% ± 8.4% and 69.9%±9.4% for the whole group. Fifty-two(74.3%)patients got negative RT-PCR result at least once. Serial monitoring of RT-PCR was available in 38 cases and 24(63.2%)patients presented with persistent negative PGR results. The achievement and continuous negative RT-PCR result was significantly related to the RFS. Conclusions Achievement of negative RT-PCR in remission is associated with favorable RFS and OS. Continuous negative RT-PCR results are associated with long-term relapse-free survival and may be considered as potentially curative. RT-PCR assay for detection of MRD should be performed regularly during post-remission period as an important prognostic factor. Chin Med J 2000; 113(10): 899-902

Objective To study the effects of high concentrations of glucose, insulin and oxidized low density lipoprotein(ox-LDL)on apoptosis in cultured bovine aortic endothelial cells(ECs). Methods For qualitative determination of EC apoptosis, acridine orange(AO)/ethidium bromide(EB)staining and DNA agarose gels electrophoresis were used. Cellular DNA fragmentation ELISA measured apoptosis by quantitating the fragmentation of 5-bromo-2'-deoxyuridine-labeled DNA. Results High concentrations of glucose(20 mmol/L, 40 mmol/L), insulin(3000 μU/ml)and ox-LDL(50μg/ml, 100 μg/ml)induced concentration-and time-dependent apoptosis in ECs. They had a synergetic effect on EC apoptosis. The combined effect of high concentration of glucose, insulin and ox-LDL was greater than any two of them; the effect of two was greater than one alone. Low concentration of insulin(30 μU/ml)decreased apoptosis in ECs induced by high concentrations of glucose(40 mmol/L), but no similar effect occurred with ox-LDL(100 μg/ml). Conclusion High ambient glucose, insulin and ox-LDL can induce excessive apoptosis in cultured ECs, and low ambient insulin can prevent EC apoptosis. Excessive EC apoptosis induced by the separate or synergetic effect of hyperglycemia, hyperinsulinemia and hyperlipidemia may be one of the reasons for loss of endothelial integrity, dysfunction of the vascular endothelium and increased plasma membrane permeability, which are all involved in the development of diabetic macrovascular complications. Chin Med J 2000; 113(l0): 903-906

Objective To investigate the cellular immune mechanism of thyroid associated ophthalmopathy(TAO)and provide a basis for treating TAO with cytokine or anti-cytokine agents, we determined whether interferon(IFN)-γ and interleukin(IL)-4, representative cytokines of Th1 and Th2 cells, may have some effect on the development and progression of TAO. Method Retroorbital fibroblasts(RF)proliferation and the synthesis of hyaluronic acid(HA)and type IV collagen were measured with liquid scintillation and radioimmunoassay. Results IFN-γ stimulated RF proliferation and HA synthesis and had a significant inhibitory effect on type IV collagen synthesis. IL-4 stimulated proliferation and type IV collagen synthesis in RF and had inhibitory effect on HA synthesis. When IFN-γ(100 U/ml)and IL-4(1 μg/L)were incubated together with RF, they antagonized their respective stimulatory or inhibitory effect on the proliferation and synthesis of HA and type IV collagen. Thyrotropin(TSH)stimulated RF proliferation and type IV collagen synthesis in a dose-dependent manner, while only at high dose(100 U/L and 200 U/L), stimulated RF to synthesize HA. Conclusions IFN-γ, a representative cytokine of Th1 cells, is responsible for the inflammatory process of TAO; whereas IL-4, a representative cytokine of Th2 cells, has some effect on the repair process. IL-4 could antagonize the inflammatory effect of IFN-γ on RF. TSH may have aggravating effect on the pathogenesis of TAO. Chin Med J 2000; 113(10): 907-910

Objective To detect the influence of antisense s-oligodeoxynucleotides(S-ODNs)of bd-2 and multidrug resistamce-associated protein(MRP)genes multidrug resistance-associated protein gene and bcl-2 antisense S-oligodeoxynucleotides on cisplatin-resistant lung adenocarcinoma cell line A549DDP which overexpresses both bcl-2 and MRP. Methods A549DPP cells were treated with sense and antisense S-ODN mediated by lipofection. Expression of MRP and bcl-2 mRNA and protein in the treated cells was measured by RT-PCR and flow cytometry(FCM), respectively. Apoptosis was identified by DNA electrophoresis and terminal deoxynucleotidyl transferase(TdT)-mediated biotin dUTP nick end-labeling(TUNEL). The degree of drug resistance of the treated cells was detected by a cell viability 3'-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tefrazolium bromide thiazolylblue(MTT)assay. Results Expression of bcl-2 and MRP significantly decreased in the cells treated with bcl-2 or/and MRP antisense S-ODN for 48h as compared to the cells untreated and sense-treated(P < 0.05). Resistance to cisplatin in the cells treated with bcl-2 or/and MRP antisense S-ODN decreased by 60.6%(6.5 times), 56.4%(7.2 times)and 71.0%(4.8 times), respectively, which paralleled the decrease of bcl-2 and MRP expression. Similarly, the resistance to etoposide and epirubicin in antisense-treated cells also reduced in parallel to decreases of the two gene expressions. The drug resistance in sense-treated cells was similar to that in untreated cells. Statistically significant dose-and concentration-dependent increases of apoptotic cells were observed in the groups exposed to 100 μmol/L cisplatin for 48 h after treatment by bcl-2 or/and MRP antisense. Conclusion Bcl-2 and MRP were at least additive and possibly synergistic in conferring drug resistance in a cisplatin-resistant lung adenocarcinoma cell line. Antisense S-ODN could attenuate drug resistance by promoting cells apoptosis, which might lead to a new treatment for patients with non-small cell lung cancers(NSCLCs)who are refractory to conventional chemotherapy. Chin Med J 2000; 113(10): 957-960