Multidrug resistant genes are highly expressed in hepatocellular carcinoma that seriousty affects the effect of chemotherapy. Screening of resistant genes from HCC cells and studying its mechanism of drug resistance will be helpful to improve the effecacy of chemotherapy for hepatocellular carcinoma. Here we described an alternative method called cyclical packaging rescue (CPR). First we constructed a retrovirus cDNA library of hepatoma cells and used it to infect fibroblasts. Then we added drugs to screen survival cells. The survival cells, stably integrated helper-free retroviral libraries, were recovered rapidly after transfection with plasmids expressing retroviral gag-pol and env genes. Through this method, retroviral RNAs were directly repackaged into new infectious virions. Recovered retroviral supernatant was then used to reinfect fresh target cells. When performed in concert with selection using functional assays, cDNAs regulating functional responses could be identified by enrichment through multiple rounds of retroviral library recovery and retransmission. Using CPR, we obtained several cDNAs. After a preliminary detection, we found Ribosomal protein S11 (RPS11), Ribosomal protein L6 (RPL6), Ribosomal protein L11 (RPL11), Ribosomal protein L24 (RPL24) possibly had drug resistant function.
Carcinoma, Hepatocellular ; genetics ; pathology ; Cell Line, Tumor ; DNA, Complementary ; Drug Resistance, Neoplasm ; genetics ; Gene Library ; Genetic Vectors ; Humans ; Liver Neoplasms ; genetics ; pathology ; Plasmids ; Retroviridae ; Ribosomal Proteins ; genetics ; metabolism ; Transfection

Background and purpose: Lysine specific demethylase 1(LSD1) is an important chromatin modifier. It epigenetically regulates gene expression pattern through chromatin modification and participates in maintenance of tumor malignant properties, such as oncogenesis, development, invasion, migration and metabolic transformation. SIRT3 (sirtuin 3) is a mitochondria localized tumor suppressor and regulates tumor metabolic transformation and oxidative stress. The correlation between LSD1 and SIRT3 has never been reported before. This study aimed to elucidate the correlation between LSD1 and SIRT3 with gene transcriptional regulation methods. Methods: RNA interference technique, co-immunoprecipitation assay(CoIP), chromatin immune-precipitation assay(ChIP) and ifrelfy luciferase activity assay were employed to elucidate the correlation between LSD1 and SIRT3 in pancreatic cancer. Results:mRNA and protein levels of SIRT3 were signiifcantly elevated in LSD1 knock-down PANC-1 cells. LSD1 interacts with PGC-1α, an important regulator of SIRT3 gene expression. LSD1 and PGC-1αoccupied the same region in SIRT3 promoter region through ChIP analysis. Luciferase activity assay validated LSD1 as a negative regulator of PGC-1αin SIRT3 gene transcriptional regulation. Conclusion:LSD1, as an important tumor promoter, negatively regulates the expression of tumor suppressor gene SIRT3, these results provide important clues for the role that LSD1 plays in aberrant metabolism and oxidative stress.

Objective To evaluate the effects on pregnancy outcome of freezing time from oocyte retrieval and thawing method for metaphase Ⅱ human oocytes vitrification. Methods From Mar 2007 to Mar 2009, the clinical outcome of 30 infertile women undergoing vitrified-thawing oocytes of in vitro fertilizationembryo transfer(IVF-ET) in the Reproductive Medical Center of the First Affiliated Hospital of Zhengzhou University was studied retrospectively, including 21 women with double fallopian tube obstruction and 9 women's husband azoospermia. All infertile women were divided into three groups, including 5 cases in group A (freezing between 4 and 5 hours from oocyte retrieval and conventional thawing method), 9 cases in group B (freezing within 2 hours from retrieval and conventional thawing method) and 16 cases in group C (freezing within 2 hours from retrieval and improved thawing method). The vitrified oocytes were preserved for 2 months to I year and thawed for Intracytoplasmic sperm injection (ICSI) and embryo transfer. The outcome of IVF and pregnancy were recorded. Results (1) The rates of oocyte survival was (65±33) % in group B and (72±23)% in group C and the rate of transfer cycle was 9/9 in group B and 16/16 in group C, which were all significantly higher than (16±17) % of oocyte survival and 1/5 of transfer cycle in group A (P = 0. 001,0. 021). However, the rate of oocyte survival and transfer cycle between group B and group C did not reach statistical difference (P > 0. 05). The rate of implantation and clinical pregnancy of (33±38) % and 9/16 in group C were significantly higher (4±11)% and 1/9 in group B (P =0. 033,0. 040).(2)The mean age of women in group C were (28.6±2.1) in oneself oocyte, (28.0±4.6) in donor oocyte and (28.1±3.4) in donor sperm. The rate of oocyte survival was (73±25) %, (88±10) % and (66±25) %. The rate of fertilization rate was (84. 6±0. 9) %, (79. 3±2. 0) % and (82. 8±15.0) %. The rate of implantation was (20. 0±44. 7) %, (33. 0±0. 1) % , (41.6±41.7) %. The rate of clinical pregnancy was 1/5 in oneself cycles,3/3 in donor oocyte cycles, 5/8 banked donor sperm cycles in group C. All above clinical parameters were not statistically different (P >0. 05). (3) In group A, one women underwent IVFET and no clinical pregnancy was observed. One women pregnancy was terminated at two months in group B.The clinical pregnancies rate of group C was 9/16, late abortion occurred in 1 woman, the other 8 women underwent term pregnancy, including 5 male infants and 4 female infants. All of infants showed normal Karyotype. Live-birth rates per warmed oocyte was 5.9% (8/135). The mean gestational weeks and birth weight of the infants were (39. 4±0. 9) weeks and (3574±569) g, respectively. Conclusions Embryo quality and clinical outcome of thawing cycles could be significantly improved when oocyte vitrification was performed within 2 hours from oocyte retrieval and improved thawing method.

Objective To investigate the relationship between apparent diffusion coefficient (ADC)value of rectal adenocarcinoma on DWI and its pathological grading.Methods The ADC values of 46 rectal adenocarcinomas were measured and compared with their histopathological grades.Results The 46 rectal adenocarcinomas included well differentiated adenocarcinomas in 14,moderate-ly differentiated ones in 20,and poorly differentiated ones in 12.The ADC values of well,moderately and poorly differentiated ade-nocarcinomas were (1.125±0.103)×10 -3 mm2/s,(1.030±0.098)×10 -3 mm2/s and (0.922±0.091)×10 -3 mm2/s,respective-ly,exhibiting a statistical difference (χ2 =1 7.35 1,P =0.000).Mann-Whitney U test showed that difference in ADC value between different histopathological grades was statistically significant.Conclusion ADC value of rectal adenocarcinoma can be used as a bio-marker for cell grading to guide treatment decision and prognosis assessment.

Objective:To investigate the effect of ultrasound combined with 4-hydroxyphenyl-retinamide (4-HPR) lipid microbubbles on type Ⅰ collagen α1 chain (COL1A1) protein expression in keloid-derived fibroblasts.Methods:In vitro cultured keloid-derived fibroblasts were divided into 3 groups: control group receiving conventional culture with incomplete Dulbecco′s modified Eagle′s medium (DMEM) , 4-HPR lipid microbubble group cultured with incomplete DMEM containing 15 mg/L 4-HPR lipid microbubbles, and ultrasound + 4-HPR lipid microbubble group cultured with incomplete DMEM containing 15 mg/L 4-HPR lipid microbubbles under ultrasound treatment. After 24-hour treatment, reverse transcription (RT) -PCR and Western blot analysis were performed to determine the mRNA and protein expression of COL1A1 in keloid-derived fibroblasts in each group. Intergroup comparison was carried out by using t test. Results:The mRNA relative expression level of COL1A1 was 1.00 ± 0.18, 0.69 ± 0.15 and 0.35 ± 0.18 in the control group, 4-HPR lipid microbubble group and ultrasound + 4-HPR lipid microbubble group respectively, and the protein relative expression level of COL1A1 was 0.93 ± 0.03, 0.74 ± 0.07 and 0.44 ± 0.06 in the above 3 groups respectively. Moreover, the mRNA and protein expression of COL1A1 was significantly lower in the 4-HPR lipid microbubble group and ultrasound + 4-HPR lipid microbubble group than in the control group ( P < 0.05 or 0.001) , and lower in the ultrasound + 4-HPR lipid microbubble group than in the 4-HPR lipid microbubble group ( P < 0.05) . Conclusion:Ultrasound combined with 4-HPR lipid microbubbles could markedly inhibit the mRNA and protein expression of COL1A1 in keloid-derived fibroblasts.

Objective To evaluate the inhibitory effect of fenretinide-loaded liposomes(4-HPR-L) on subcutaneous transplanted malignant melanomas in nude mice.Methods A film-ultrasonic dispersion method was used to prepare 4-HPR-L.BALB/c nude mice were subcutaneously inoculated with A375 melanoma cells in the right axillary fossae to establish malignant melanoma-bearing nude mouse models.Ten nude mouse models were randomly and equally divided into 2 groups to be injected with near-infrared fluorescent cell membrane label (DiR) solution or DiR liposomes (DiR-L)at the same concentration in the caudal vein,and a live imaging system was used to observe the distribution of DiR or DiR-L in nude mice at 6,12,24 hours after the injection.Another 30 nude mice were randomly and equally divided into 3 groups to be injected with 5％ (mass fraction) glucose solution at a single-dose of 0.2 ml (control group),25 mg/kg 4-HPR solution (4-HPR group)and 25 mg/kg 4-HPR-L solution (4-HPR-L group) respectively on days 8,10,12,14,16,18,20 and 22 after the inoculation with A375 cells.The mouse body weight and tumor volume were dynamically monitored in the above groups after the injection,and the survival situation was observed.The nude mice were sacrificed on day 2 after the final injection,and the heart,liver,spleen,lung,kidney and tumor tissues were resected.These tissues were subjected to hematoxylin-eosin staining and immunohistochemical staining to observe the metastasis of melanoma in mice,and terminal deoxyribonucleotidyl transferase-mediated nick end labelling was performed to detect the apoptosis in tumor cells.One-way analysis of variance and independent-sample t test were used to analyze measurement data.Results The live imaging system showed that DiR-L could be retained in melanoma for a long time,and strong fluorescence of DiR-L could be still observed in the tumors at 24 hours after injections.Quantitative fluorescence analysis revealed that the fluorescence intensity of DiR-L (22.85 ± 1.66) was significantly higher than that of DiR in tumor tissues (8.45 ± 0.97,t =12.957,P ＜ 0.01).Compared with the control group and 4-HPR group,the resected tumor weight on day 2 after the final injection was significantly decreased in the 4-HPR-L group (F =27.055,t =4.768,6.640,respectively,both P ＜ 0.05).Hematoxylineosin staining showed that liver metastasis occurred in 2 nude mice in the 4-HPR-L group,but in all the nude mice in the control group and 4-HPR group.All the nude mice in the 4-HPR-L group died within 76 days after inoculation,and the mice in the control group and 4-HPR group all died within 56 and 59 days respectively after inoculation.There were significant differences in the apoptotic index among the control group (12.14‰ ± 1.33‰),4-HPR group (67.17‰± 15.18‰) and 4-HPR-L group (152.73‰ ± 11.27‰;F =167.588,P ＜ 0.05),and the apoptotic index was significantly higher in the 4-HPR-L group than in the control group and 4-HPR group (t =18.162,11.075 respectively,both P ＜ 0.05).Conclusion 4-HPR-L can effectively inhibit the growth of subcutaneous melanoma in nude mice and metastasis of melanoma cells,and prolong the survival duration of nude mice.

Background and purpose:Lower expression of E-cadherin is associated with metastasis of cancer cells, however, the correlation between E-cadherin and glucose metabolism has seldom been reported. This article studied the correlation between E-cadherin and glycolysis effect in PANC-1 cells.Methods:Through treatment of transforming growth factor β (TGF-β) in PANC-1 cells to decrease E-cadherin expression, knock-down the gene of E-cadherin interaction protein β-catenin, and overexpressing of E-cadherin, the effects of E-cadherin on the glucose uptake and lactate production ability and on the expression of key glycolytic genes were assessed.Results:E-cadherin negatively regulated the glycolytic effect of PANC-1 cells by inhibiting glucose uptake and lactate production (P<0.05). Moreover, E-cadherin interacting partner β-catenin signiifcantly promoted glucose metabolism transformation in PANC-1 cells (P<0.05). Moreover, key glycolysis regulator sirtuin 3 (SIRT3) could lower E-cadherin expression.Conclusion:Lower expression of E-cadherin induced the transformation of glucose metabolism transformation in PANC-1 cells and manipulation of E-cadherin expression level could change the glycolysis effect. Moreover, through maneuver glycolysis process could inhibit high metastatic potential of pancreatic cancer cells.

Objective:To evaluate the role of lactate dehydrogenase in diabetic neuropathic pain (DNP) and the relationship with peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in mice.Methods:SPF-grade healthy male C57BL/6J mice, aged 6-8 weeks, weighing 18-22 g, were used to establish diabetes mellitus model by intraperitoneal injection of streptozotocin (STZ) 120 mg/kg. Twenty-four mice with diabetes mellitus were divided into 2 groups ( n=12 each) using a random number table method: DNP group and DNP + oxamate group (OXA group). Another 12 SPF-grade healthy male C57BL/6J mice were selected as control group (C group). In OXA group, oxamate 750 mg/kg was intraperitoneally injected once a day for 28 consecutive days. The equal volume of normal saline was given instead in C group and DNP group. The mechanical paw withdrawal threshold (MWT), blood glucose and body weight were measured at 3 days before STZ injection and at 1, 2, 3 and 4 weeks after STZ injection (T 0-4). After the last behavioural test was completed, blood samples were collected from the posterior orbits of anesthetized mice for determination of serum lactate concentrations. The animals were then sacrificed and the tissues from the prefrontal cortex of the brain were taken for determination of lactate content, mitochondrial membrane potential (by the JC-1), content of reactive oxygen species (ROS) (using dihydroethidium probes), and level of histone lactylation and expression of PGC-1α (by Western blot). Results:Compared with C group, the MWT was significantly decreased at T 2-4, the serum lactate concentrations, contents of lactate and ROS and level of histone lactylation were increased, the mitochondrial membrane potential was decreased, and the expression of PGC-1α was down-regulated in DNP and OXA groups ( P<0.05). Compared with DNP group, no significant change was found in blood glucose and body weight ( P>0.05), the MWT was significantly increased at T 2-4, the serum lactate concentrations, contents of lactate and ROS and level of histone lactylation were decreased, the mitochondrial membrane potential was increased, and the expression of PGC-1α was up-regulated in OXA group ( P<0.05). Conclusions:Lactate dehydrogenase promotes the development of DNP, and the mechanism is related to promotion of increase in histone lactfication and down-regulation of PGC-1α expression in the prefrontal cortex of mice.

Lymphoedema of lower extremities,chronic and progressive,will severely deteriorate the quality of life of patients as it progresses.Thus,early diagnosis and treatment to delay the progress of the disease is conducive to improving the prognosis of patients.At present,common techniques for the diagnosis of lower limb lymphedema,whose advantages and disadvantages vary,cannot be applied to individual case comprehensively.CEUS has the advantages of non-invasion,convenience,real-time,and good repeatability for this disease.CEUS can enhance the image of lymph in lymphatics,and has a high sensitivity to superficial lymphatics,gradually applied in lymphedema of lower limbs.This article reviews the application of CEUS in lower limb lymphedema.