2004年 · 第117卷第06期

Background Mutation and expression change of p21WAF1/CIP1 may play a role in the growth of osteosarcoma. This study was to investigate the expression of the p21WAF1/CIP1 gene in human osteosarcoma, p21WAF1/CIP1 gene DNA sequence change and their relationships with the phenotype and clinical prognosis. Methods p21WAF1/CIP1 gene in 10 normal people and the tumours of 45 osteosarcoma patients were examined using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) with silver staining. The PCR product with an abnormal strand was sequenced directly. The p21WAF1/CIP1 gene mRNA and P21 protein of 45 cases of osteosarcoma were investigated by using in situ hybridization and immunohistochemistry, respectively. Results The occurrence of P21 protein in osteosarcoma was 17. 78% (8/45), and p21WAF1/CIP1 mRNA expression in osteosarcoma was 42.22% (19/45). The p21WAF1/CIP1 gene DNA sequencing of amplified production showed that in p21WAF1/CIP1 gene exon 3 of 36 cases of human osteosarcoma, there were 17 cases (47. 22%) with C→T at position 609; 10 normal blood samples' DNA sequence analysis yielded 8 cases (80.00%) with C→T at the same position. Conclusions Along with the increase of malignancy, the expression of p21WAF1/CIP1 mRNA and P21 protein in osteosarcoma tends to decrease. It is uncommon for the p21WAF1/CIP1 gene mutation to occur in human osteosarcoma. As a result, the possible existence of tumour subtypes of p21WAF1/CIP1 gene mutation should be investigated. Our research leads to the location of p21WAF1/CIP1 gene polymorphism of Chinese osteosarcoma patients, which can provide a basis for further research. Chin Med J 2004; 117(6):936-940

Background A living fetus within the maternal uterus provides an example of allogene tolerance in mammals. Poria cocos Wolf is the main component of many Chinese medicinal combination drugs that have therapeutic effects on recurrent spontaneous abortion and that can maintain pregnancy until delivery. It was hypothesized that this herbal medicine can also prolong allograft survival after organ transplantation. Here, in an in vivo study, we report the anti-rejection effect of the ethanol extract of Poria cocos Wolf (EEPCW) in rats after cardiac allograft implantation. Methods Ten normal rats were healthy controls. Eighty rats receiving homologous heart transplants were divided into 4 groups of 20 rats each based on type of treatment: olive oil 8 ml • kg-1 • d-1, EEPCW 25 mg • kg-1 • d-1, EEPCW 50 mg • kg-1 • d-1 or cyclosporin A 5mg • kg-1 • d-1. Allograft survival was observed in 10 rats from each group. On the seventh day post transplantation, pathological lesions and percentages of CD3+, CD4+, and CD8+ lymphocytes and the CD4+/CD8+ ratio in peripheral blood were assessed in another 10 rats from each group and in 10 normal rats. Results The survival time of donor hearts in the two EEPCW groups was significantly prolonged, to (15. 9 ±2. 4) days and (30. 0 ±0. 0) days, respectively, compared with (6. 7 ±0. 8) days in the control group. Pathological lesions in the two EEPCW groups were also less severe, and the percentages of CD3+, CD4+, and CD8+ lymphocytes and CD4+/CD8+ ratio were significantly lower in the EEPCW groups. Conclusions Acute rejection of heart transplants and cellular immune reaction can be effectively suppressed using the EEPCW. Taking advantage of novel immunosuppressants derived from Chinese medicinal herbs used to treat abnormal pregnancy provides a hopeful road for future research and treatment in organ transplantation. Chin Med J 2004; 117(6):932-935

Background Oligosaccharides in human milk may protect infants by improving the intestinal microflora and fermentation. This study was to investigate effects of infant formula milk consisting of galacto-oligosaccharide (GOS) on intestinal microbial populations and the fermentation characteristics in term infants in comparison with that of human milk. Methods The test formula (Frisolac H, Friesland, Netherland) was supplemented with GOS at a concentration of 0. 24 g/dl. Human milk and another formula without oligosaccharides (Frisolac H, Friesland, Netherland) were used as positive and negative control respectively. Growth, stool characteristics, and side effects of the recruited infants were recorded after 3 and 6 months' follow-up, and the fecal species were collected for the analysis of intestinal micro-flora, short chain fatty acid (SCFA) and pH. Results At the end of 3- and 6-month feeding period, intestinal Bifidobacteria and Lactobacilli were significantly increased in infants fed with GOS supplemented formula and human milk when compared with infants fed with negative control formula; however, there was no statistically significant difference between GOS supplemented formula and human milk groups. Stool characteristics were influenced by the supplement and main fecal SCFA (acetic), and stool frequency were significantly increased in infants fed with GOS supplemented formula and human milk, while the fecal pH was significantly decreased as compared with that of negative control (P <0. 05). Supplementation had no influence on incidence of side effects (including crying, regurgitation and vomiting). Conclusions Supplementing infant formula with GOS at a concentration of 0. 24 g/dl stimulates the growth of Bifidobacteria and Lactobacilli in the intestine and stool characteristics are similar to in term infants fed with human milk. Chin Med J 2004; 117(6):927-931

Background We asssessed whether the CaNa2 EDTA could improve the accumulation of protoporphyrin Ⅸ (PpⅨ) and photosensitisation in HEp-2 cells as well as the depth of treatment of skin cancers on the topical 5-Aminolaevulinic acid (5-ALA) PDT. Methods HEp-2 cells were incubated with 5-ALA (0-1 mmol/L) and CaNa2 EDTA (0-1 mmol/L) for 4 hours, intracellular protoporphyrin Ⅸ content was quantified by extraction, and cell viability was assessed by use of the methyl-tetrazolium (MTT) assay four hours after exposure to light. In comparison with the pictures before and after treatment, depth of treatment could be determined using a Acuson Sequioa 512 phase-array system in paired experiments. Results PpⅨ accumulation increased with increasing extracellular concentrations of ALA (0-1 mmol/L). Adding 1 mmol/L of CaNa2EDTA increased 30% PpⅨ accumulation over the same period of incubation in the concentration of 1 mmol/L ALA. Significant difference was observed between the 5-ALA alone group and 5-ALA combined CaNa2 EDTA group in the PpⅨ accumulation (P<0. 01). Cell viability after exposure to light decreased with adding CaNa2 EDTA, a statistical difference in a same fluence above 1. 2 J/cm2 between two groups was demonstrated (P <0. 05, P <0. 01 respectively). Depth of treatment of skin cancers were increased in CaNa2 EDTA-treated group. conclusion CaNa2 EDTA could improve the PpⅨ accumulation and photosensitisation in HEp-2 cells. Clinically, CaNa2 EDTA could increase the depth of treatment in the cutaneous cancers. Chin Med J 2004; 117(6):922-926

Background Batroxobin (BX), a serine protease used in defibrinogenation and thrombolysis, also has an effect on c-fos gene and growth factor. This study attempted to determine the effects of BX on the proliferation of vascular smooth muscle cells (VSMCs) and calcium metabolism. Methods VSMCs were treated with BX at concentrations of 0. 1, 0. 3, or 1. 0 mmol/L and cell numbers were determined at 0, 24, 48, and 72 hours. Intracellular calcium concentration ([Ca2+]i) was measured using direct fluorescence methods. Results BX was found to suppress proliferation of VSMCs in a dose-dependent fashion with inhibition rates of 18% and 31 % by 48 and 72 hours, respectively. In addition, BX decreases basal [Ca2+]i significantly. The basal level in untreated cells was 162. 7 ±33. 8 nmol/L, and decreased to 131. 5 ± 27.7 nmol/L, 128.3 ±28.5 nmol/L, and 125. 6 ±34. 3 nmol/L with the three concentrations of BX, respectively. Noradrenaline (NE)-induced [Ca2+]i stimulation was also attenuated by BX (0. 1 mmol/L BX, 20% ±8% inhibition; 0. 3 mmol/L BX, 54% ±11% inhibition; 1.0 mmol/L BX, 62% ± 15% inhibition). The ability of NE to stimulate [Ca2+]i was attenuated in cultures in Ca2+-free medium, as was the ability of BX to blunt NE-induced stimulation. Conclusion These findings demonstrate that BX can effectively inhibit proliferation of VSMCs, probably by blocking the release and uptake of Ca2+, thus influencing [Ca2+]i. Chin Med J 2004; 117(6):917-921

Background The contractility of hepatic stellate cells (HSCs) may play an important role in the pathogenesis of cirrhosis with portal hypertension. The aim of this study was to research the effects of octreotide, an analogue of somatostatin, on intracellular Ca2+ and on the expression of L-type voltage-operated calcium channels (L-VOCCs) in activated HSCs, and to try to survey the use of octreotide in treatment and prevention of cirrhosis with portal hypertension complications. Methods HSC-T6, an activated HSCs line, was plated on small glass coverslips in 35-mm culture dishes at a density of 1 × 105/ml, and incubated in DMEM media for 24 hours. After the cells were loaded with Fluo-3/AM, intracellular Ca2+ was measured by Laser Scanning Confocal Microscopy (LSCM). The dynamic changes in activated HSCs of intracellular Ca2+, stimulated by octreotide, endothelin-1, and KCI, respectively, were also determined by LSCM. Each experiment was repeated six times. L-VOCC expression in HSCs was estimated by immunocytochemistry. Results After octreotide stimulation, a signifcant decrease in the intracellular Ca2+ of activated HSCs was observed. However, octreotide did not inhibit the increases in intracellular Ca2+ after stimulation by KCI and endothelin-1. Moreover, octreotide did not significantly affect L-VOCC expression. These results suggest that neither L-VOCC nor endothelin-1 receptors in activated HSCs are inhibited by octreotide. Conclusions Octreotide may decrease portal hypertension and intrahepatic vascular tension by inhibiting activated HSCs contractility through decreases in intracellular Ca2+. The somatostatin receptors in activated HSCs may be inhibited by octreotide. Chin Med J 2004; 117(6):913-916

Background Repeated attacks of bronchial asthma lead to different degrees of airway remodeling, the mechanism of which is not yet clear. Some evidences indicate that it is related to the excessive expression of some growth promotion factors. Angiotensin Ⅱ is a polypeptide that may be involved in airway remodeling. To evaluate its role in airway remodeling in asthma, we observed the effects of an angiotensin Ⅱ type 1 receptor antagonist (valsartan) on the expression of collagen Ⅲ, collagen V, and transforming growth factor β1 (TGF-β1) mRNA and protein in the airway walls of sensitized rats. Methods Forty Wistar rats were randomly divided into 5 groups: control group, sensitized group, and valsartan groups 1, 2, and 3. The rats in the sensitized group and in valsartan groups 1, 2, and 3 were sensitized and challenged with ovalbumin. Rats in control group were sensitized and challenged with 0. 9% NaCI. Rats from valsartan groups 1, 2, and 3 were drenched with valsartan (10 μg, 20 μg, or 30 μg, respectively) at the time of the ovalbumin challenges. The expression of collagen Ⅲ, collagen V, and TGF-β1 protein were detected using immunohistochemical method in combination with image analysis methods. The expression of TGF-β1 mRNA was detected by in situ hybridization. Results The expression in the airways of collagen Ⅲ and collagen V was significantly higher in rats from the sensitized group (7. 73 ±0. 81, 1. 34 ±0. 28) and from valsartan groups 1, 2, and 3 (5. 73 ± 0. 64, 1. 13 ±0. 15; 4. 96 ±0. 51, 0. 98 ±0. 08; 4. 43 ±0. 35, 0. 93 ±0. 06, respectively) than those in the control group (2. 65 ±0.38, 0. 67 ±0. 08, P < 0. 05). In addition, collagen levels were significantly lower in valsartan groups 1, 2, and 3 than those from the sensitized group (P <0. 05). The expression of TGF-β1 mRNA and protein in the airways was significantly higher in rats from the sensitized group (20. 49% ±3.46%, 29. 73% ± 3. 25%) and from valsartan groups 1, 2, and 3 (16. 47% ±1.94%, 19.41% ±1.87%; 14. 38% ± 1. 58%, 18. 29% ± 1. 43%; 12. 96% ± 1. 73%, 18. 63% ± 1. 11%, respectively) than that from the control group (7. 84% ±1.61%, 5. 63% ± 1. 07%, P<0. 05). TGF-β1 mRNA and protein levels were significantly lower in valsartan groups 1, 2, and 3 than that in the sensitized group (P<0. 05). Conclusions Angiotensin Ⅱ receptor antagonist valsartan can suppress synthesis of collagen Ⅲ and collagen V by downregulating TGF-β1 mRNA and protein expression. Valsartan can decrease airway remodeling and could play a role in asthma therapy. Chin Med J 2004; 117(6):908-912

Background There is a higher rate of cytomegalovirus (CMV) reactivation in patients with multiple myeloma after an autologous stem cell transplantation, but no attention has been given thus far to a possible pathogenetic interplay between CMV and multiple myeloma. CMV can infect many kinds of cells, and CMV infection has been shown to inhibit apoptotic responses in several cell systems. In this study the authors investigated the alterations in apoptosis in the multiple myeloma cell line KM3 after infection with CMV and proposed a possible mechanism. Methods KM3 cells were infected with 100, 10, or 1 TCID50 of CMV and then cultured in serum-free RPMI 1640. An RT-PCR-based assay was used to detect mRNA expression of CMV-IE, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and IL-6 in CMV-infected and mock-infected cells. Flow cytometry was used to detect apoptotic cells. CMV particles and apoptotic cells were also examined with an electron microscope. Results CMV-infected KM3 cells clearly expressed immediate early (IE) antigen mRNA when compared to uninfected cells, and there were fewer apoptotic cells among cells treated with 100 or 10 TCID50 of CMV after culturing in serum-free RPMI 1640. CMV particles were observable in infected cells under an electron microscope. Expression of IL-6 mRNA increased after infection. Conclusion CMV can infect the multiple myeloma cell line KM3, inhibit the apoptotic response in these cells after apoptosis induction in serum-free culture, and increase the expression of IL-6 mRNA. Chin Med J 2004; 117(6):903-907

Background Nurr1 is a member of the nuclear receptor superfamily of transcription factors. The objective of the present study was to identify novel splicing variants of the gene in neuronal and non-neuronal tissues and determine their functions. Methods Reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to screen for Nurr1 splice variants in the adult human central nervous system (CNS) and in other tissues such as lymphocytes, and liver, muscle, and kidney cells. Functional assays of the variants were performed by measuring Nurr1 response element (NuRE) transcriptional activity in vitro. Results In this study, the authors identified a novel splicing variant of Nurr1 within exon 5, found in multiple adult human tissues, including lymphocytes, and liver, muscle, and kidney cells, but not in the brain or spinal cord. Sequencing analysis showed the variant has a 75 bp deletion between nucleotides 1402 and 1476. A functional assay of the Nurr1-c splicing variant, performed by measuring NuRE transcriptional activity in vitro, detected a 39% lower level of luciferase (LUC) activity (P < 0.05). Conclusion A novel splicing variant of Nurr1 exists in human non-neuronal tissues and functional assays suggest that the variant may act as an alternate transcription regulator. Chin Med J 2004; 117(6):899-902

Background Retroviral vectors have been widely used to introduce foreign into various target cells in vitro, thus showing relatively high systemic delivery efficiency of various transgene products. The authors investigated the stability and efficiency of skeletal muscle-specific hybrid retroviral vectors in expression of human factor Ⅸ (FIX) in vitro and iv vivo. Methods FIX cDNA in LIXSN vector was replaced with a FIX minigene containing splicing donor and splicing acceptor sequence of first intron of human FIX gene. Two copies of muscle creatine kinase enhancer (MCK, Me2) were inserted in forward or reverse orientation at Nhel site of 3, long terminal repeat (LTR), resulting in two hybrid vectors, which were designated as LMe2IXm2SN (F) and LMe2IXm2SN(R), respectively. The vectors were tested in vitro and in vivo for stability and muscle-specificity of factor Ⅸ expression with SCID mice. Results Muscle cells carrying vector with Me2 expressed significantly higher levels of FIX (up to 1800 ng/106. 24h) than those without Me2, thus suggesting that Me2 could specifically increase expression level of FIX in muscle cells. Myoblasts transduced with LMe2IXm2SN(R) produced much less FIX in vivo in SCID mice than LMe2IXm2SN (F). One or two copies of Me2 sequence were deleted in myoblasts transduced with LMe2IXm2SN(R) without changing the orientation of Me2. Conclusions LTR inserted with MCK enhancers can specifically increase human FIX expression in skeletal muscle cells in vitro and in vivo, and MCK enhancer should be positioned in the same orientation as that of LTR promoter. Chin Med J 2004; 117(6):893-898