Real-time quantitative detection of E2A-PBX1 fusion gene in children with acute lymphoblastic leukemia and its clinical application in minimal residual disease monitoring / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To establish a real-time reverse transcription-polymerase chain reaction (RT-PCR) for quantitative detection of E2A-PBX1 fusion gene mRNA in acute lymphoblastic leukemia (ALL) children and to explore its clinical significance in minimal residual disease monitoring and prognosis evaluation.</p><p><b>METHODS</b>Real-time RT-PCR was used to quantitatively detect the mRNA expression of E2A-PBX1 gene in 11 newly diagnosed ALL patients at diagnosis (11 cases), complete remission (11 cases) and periods of relapse (3 cases). Ten children with normal bone marrow cell morphology and without hematopathy or tumor diseases were used as the control group.</p><p><b>RESULTS</b>The median expression levels of E2A-PBX1 fusion gene in the ALL group at diagnosis and the relapse group were significantly higher than in the control and complete remission groups (P<0.01). Compared with E2A-PBX1 negative patients on day 33 during induction of remission, the recurrence rate increased and disease free survival rate at 3 year decreased significantly in E2A-PBX1 positive patients decreased (P<0.05).</p><p><b>CONCLUSIONS</b>Measurement of E2A-PBX1 levels by real-time RT-PCR is useful for monitoing minimal residual disease, prediction of relapse and individual treatment. The expression level of E2A-PBX1 gene on day 33 during induction of remission can be used for prognosis evaluation.</p>

Detection of WT1 gene in acute myeloid leukemia children by real-time fluorescent quantitative RT-PCR and its clinical significance / 中国实验血液学杂志

Objective of this study was to establish a SYBR Green Ireal-time reverse transcription-polymerase chain reaction (RT-PCR) for quantitative detection of WT1 gene mRNA in children with acute myeloid leukemia (AML) and investigate its clinical significance. SYBR Green Ireal-time RT-PCR was used to quantitatively detect the mRNA expression of WT1 gene in 30 newly diagnosed AML patients, 12 cases of remission (30), 18 relapsed patients and 30 cases of normal bone marrow cell morphology, and dynamically to detect the expression of WT1 gene in 20 newly diagnosed AML children. ABL served as internal reference gene, and the 2(-ΔΔct) method was used to calculate the relative expression. The results showed that (1) the expression of WT1 gene in newly diagnosed AML children was higher than that of the normal controls and the patients with remission (p < 0.001); there were no significant difference of WT1 gene expression between AML patients with remission and normal controls (p > 0.05), which were same as in relapsed patients and newly diagnosed patients (p > 0.05); (2) WT1 gene in 20 newly diagnosed AML children highly expressed before the children were initially treated, decreased when they were complete remission, then expression increased again when their AML relapsed. The WT1 gene expression level began to rise in 5 cases before clinical relapse at 5 - 7 months; (3) the complete remission rate (CR) and 3 year overall survival (OS) did not show significant difference between the WT1-positive group and negative group when dynamically monitoring WT1 gene expression of 20 newly diagnosed children with AML. 3-year OS of WT1-positive group at the 22 - 30 days after initial treatment was significantly lower than that of the negative group (p < 0.05). It is concluded that SYBR Green Ireal-time RT-PCR is a rapid, efficient, sensitive and specific method. WT1 gene in AML childhood plays a role of cancer-promoting. The change of WT1 gene expression level contributes to evaluate the therapeutic efficacy, detect the minimal residual diseases and analyze the prognosis.

Reversal of multidrug resistance in hepatocellular cell line HepG2R by mdr1-antisense RNA / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate whether multidrug resistance gene 1(mdr1) could reverse multidrug resistance (MDR) in HepG2R cells.</p><p><b>METHODS</b>An adenovirus vector, Adeno-asmdr, containing the antisense RNA driven by AFP promoter, was construct. Adeno-EGFP was transfected into HepG2, an AFP producing cell line, L02, a normal human liver cell line, and HeLa, a human cervical cancer cell line, the EGFP transcription level was detected by RT-PCR. Adeno-asmdr was transfected into HepG2R cells, the expression of P-gp170 was detected by western blotting, apoptosis was detected using TUNEL and flow cytometry, cell cycle was analyzed by flow cytometry.</p><p><b>RESULTS</b>EGFP was highly expressed in HepG2 cells, however, its expression in L02 or HeLa cells was very weak. Western blot showed that the P-gp170 was marked down-regulated 48h after transfection with Adeno-asmdr, and the expression of P-gp170 was detectable at least 7d post-transfection. Compared with control cells, Adeno-asmdr transfected HepG2R cells were more sensitive to different chemicals, as indicated by TUNEL staining and flow cytometry. Chemical treatment arrested the cells in S and G0/M phase.</p><p><b>CONCLUSION</b>The recombinant adenoviral vector, Adeno-asmdr, can block the expression of mdr1, and reverse MDR in HepG2R cells.</p>

Reversal of adriamycin resistance of hepatocellular carcinoma by targeting it with recombined adenovirus carrying antisense multidrug resistance gene 1 RNA / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate if an adenovirus vector carrying antisense multidrug resistance gene 1 (MDR1) could reverse multidrug resistance (MDR) of HepG2/ adriamycin (ADM) cells in tumors transplanted in athymic mice.</p><p><b>METHODS</b>An adenovirus vector carrying AFP promoter and antisense MDR1 was constructed. HepG2 MDR cells (HepG2/ADM) were induced by graded resistance to ADM and were subcutaneously inoculated into athymic mice to construct the transplanted tumor. After adeno-asmdr1 was injected, the volume of the transplanted tumor and the apoptotic body in the xenograft tumor cells were observed and reverse transcriptase polymerase chain reaction was employed to investigate the expression of the mdr1-mRNA from the mouse transplanted tumor cells.</p><p><b>RESULTS</b>Following injection with adeno-asmdr1, the tumor volumes in this mice group did not increase. However the tumor volume in the PBS plus ADM group did increase significantly (P less than 0.05). In the tumor xenograft cells, mdr1 mRNA in the xenografts was assessed by RT-PCR and found to be reduced at week 1, and at week 4 in the ADM+asmdr1 group, but it was stable in the ADM group. It was only 20% in the ADM+asmdr1 group compared to the ADM group at the 4th week. Evidence of apoptosis was observed in the tumor xenograft cells treated with adeno-asmdr1, but there was rarely any apoptosis in the group treated with ADM and PBS.</p><p><b>CONCLUSION</b>Adenovirus carrying antisense mdr1 RNA can partially reverse the MDR of HepG2/ADM cells and inhibit tumor growth by down-regulating mdr1 mRNA resulting in tumor cell apoptosis.</p>

Study on ligustrazine in reversing multidrug resistance of HepG2/ADM cell in vitro / 中国中药杂志

<p><b>OBJECTIVE</b>To study the reverse effect of ligustrazine (TMP) on HepG2/ADM, a herd of hepatocellular carcinoma cell, multidrug resistance (MDR) and the influence of P-gp170 expression.</p><p><b>METHOD</b>The reverse effect of ligustrazine on HepG2/ADM cell was observed, with the methods of cell culture, MTT's analyze, RT-PCR and Flow cytometric, etc.</p><p><b>RESULT</b>Ligustrazine could make MDR of cell line of HepG2/ADM reduce the expression of P-gp170, enhance the density of adriamycin in cell and increase the adriamycin's cytotoxicity. With the Flow cytometric, the results of RT-PCR showed the transcriptional activity of the MDR1 decreased.</p><p><b>CONCLUSION</b>Ligustrazine can reverse MDR of HCC cell line of HepG2/ADM and has prospect in clinical use.</p>

In vivo expression of lipopolysaccharide binding protein and its gene induced by endotoxin / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the expression of lipopolysaccharide binding protein (LBP) and its gene in rats with endotoxemia and explore the role of LBP in the response of host to endotoxin.</p><p><b>METHODS</b>Thirty Wistar rats were divided randomly into five groups: the normal group and the endotoxemia groups (1, 3, 6, 12 hours after LPS injection, respectively). The level of plasma endotoxin was determined by the Limulus Amebocyte Lysate assay. The expression of LBP mRNA in hepatic tissue was examined by reverse transcription polymerase chain reaction (RT-PCR). Plasma levels of LBP, tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 were measured by enzyme-linked immunosorbent assay (ELISA). Morphologic changes of hepatic tissue were observed under transmission electron microscope.</p><p><b>RESULTS</b>The level of plasma endotoxin peaked at 1 h after LPS injection, then declined, but was still higher than that of the normal group at 12 h; intrahepatic expression of LBP mRNA and plasma LBP increased with time after LPS stimulation; TNF-alpha and IL-6 in plasma increased with upregulation of LBP expression; there were significant differences between the normal group and endotoxemia groups (P<0.05). Activation of Kupffer cells and injury of hepatocytes could be seen in rats with endotoxemia.</p><p><b>CONCLUSIONS</b>LPS can upregulate the intrahepatic expression of LBP mRNA and increase the plasma LBP level. Under certain conditions, LBP may enhance the sensitivity of host to the toxic effects of LPS.</p>

The role of cyclooxygenase 2 and prostaglandin I2 in the development of portal hypertensive gastropathy / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To study the role of cyclooxygenase 2 (COX 2) and prostaglandin I2 (PGI2) in the development of portal hypertensive gastropathy (PHG).</p><p><b>METHODS</b>Forty Wistar rats were divided into surgery group (32) and control group (8). Partial portal vein ligation method was used to narrow the sectional area of portal vein to establish the experimental model of PHG in surgery group rats. Then they were divided into four groups (8 rats in each). The free pressure of portal vein was determined at the 1st, 2nd, 3rd, 4th weeks after the operation, and 8 rats were killed to observe the pathological change of gastric mucosa. The levels of 6-keto-PGF1 alpha, a stable metabolite of PGI2, were determined by radioimmunoassay in gastric mucosa homogenate and the blood of portal vein. The expression of COX 2 in gastric mucosa was determined by immunohistochemistry.</p><p><b>RESULTS</b>The free pressure of portal vein increased rapidly after partial portal vein ligation and maintained a high stable level after 1 week. They were (2.40+/-0.15) kPa, (2.38+/-0.17) kPa, (2.52+/-0.21) kPa, and (2.46+/-0.17) kPa at the 1st, 2nd, 3rd, and 4th weeks after partial portal vein ligation, while it was (0.90+/-0.16) kPa in control group (t>or=17.356, P<0.05). The gastric mucosa appeared pale, edema, hyperaemia, surface erosion, punctate hemorrhage and these lesions were more apparent with the time after the operation. The pathological examination showed that the gastric mucosa and submucosa thickened. The vessels of gastric mucosa and submucosa expanded and increased. There were lymphocytes and neutrophils infiltration around the vessels in the gastric mucosa and submucosa. The 6-keto-PGF1 alpha levels in gastric mucosa and the blood of portal vein increased rapidly and maintained a high level after partial portal vein ligation,which were higher than those in control group (104.52pg/ml+/-25.11pg/ml vs 73.62pg/ml+/- 20.33pg/ml, t=2.710, P<0.05; 180.21pg /ml+/-37.56pg /ml vs 142.11pg /ml+/-31.51pg /ml, t=2.198, P<0.05). The results of immunohistochemistry showed that the intensity and degree of the COX 2 staining in gastric tissue increased at the 1st, 2nd, 3rd, 4th weeks after partial portal vein ligation, while the COX 2 in control group rats was negative.</p><p><b>CONCLUSIONS</b>The expression of COX 2 and PGI2 in gastric tissue increased in portal hypertension. PGI2 as an inflammatory medium, damages the gastric mucosa by expanding vessels and other mechanisms in portal hypertension. It may be one of the important factors contributing to the development of PHG.</p>