Objective To establish effective method for large-scale purification of islet cells from pig pan-cress. Methods Pig pancreas tissue was digested with collagenase P followed by purification in a HCA-Fi-coil dis continuous gradient using Cobe2991 cell separator. After isolation, the islet cell yield and purity were evaluated with light microscope with DTZ staining, and the islet function assessed by insulin release as-say in vitro. Results The number of the islets coll ected from each pancreas averaged (275 000±20 895)islet equivalents (IEQ) before purification, and (230 350±26 679) IEQ after the purification with discon-tinuous gradient centrifugation. From each gram of the pancreatic tissue, (2710±229) IEQ were obtained with an average purity of (50.2±1.95) %. The purified islets responded well to high-concentration (16.7 mmol/L) glucose stimulation with a 4. 74-fold increase of insulin secretion over the basal level (3.3 mmol/L, P <0.001). Conclusion The established method can be applicable for large-scale purifi-cation of fully functional islet cells from pig pancreas.

BACKGROUNDSmoking plays an important role in the development of carcinoma of human lung,but the mechanism is unknown.The objective of this study is to investigate the mutation in the coding region of mitochondrial DNA(mtDNA) in patients with squamous cell carcinoma of the lung and to explore its significance in carcinogenesis.

METHODSWhite blood cells,pericarcinomatous tissues and cancer tissues were obtained from 15 cases of lung squamous cell carcinoma and mtDNA was extracted by one step method.The part of coding region fragments was amplified by PCR.Mutations were determined by DNA sequencing.

RESULTSFifteen pairs of matched pericarcinomatous tissues and cancer tissues were screened for mutation in coding regions,18 mutations were detected in the coding region of mtDNA in 11 patients,of which 10 cases were smokers.

CONCLUSIONSThe mutations of the coding region of mtDNA in squamous cell carcinoma of the lung may be correlative with smoking.

BACKGROUNDAs a new member of inhibitor of apoptosis protein(IAP) family,Livin,especially Livin α,is known to be involved in occurrence and development of lung cancer.Livin is an important mechanism of chemotherapy resistance of lung cancer cell.The aim of this study is to set up Livin isoform(α & β)-specific gene silencing system in SPC-A1 cells by gene transfection and RNA interference(RNAi),and to explore the different functions and value of the isoforms in enhancing chemosensitivity of SPC-A1 cells.

METHODSLivinα+β,Livinα and Livinβ specific siRNA were expressed stably in SPC-A1 cells,respectively.MTT was performed to study sensitivity of the cells to chemotherapy drugs.In vivo experiment was performed to test sensitivity of mouse bearing tumor to cisplatin after gene silencing of Livin.

RESULTSAfter silencing of Livinα+β,Livinα and Livinβ genes,sensitivity of SPC-A1 cells to many chemotherapy drugs(including cisplatin,carboplatin,cyclophosphamide and adriblastine) was markedly increased(P < 0.05).Among them,gene silencing of Livinα+β showed the strongest enhancement effect on chemosensitivity of SPC-A1 cells(P < 0.01).Animal experiment showed that tumor inhibition rate of pSilencer-Livinα+β,pSilencer-Livinα and pSilencer-Livinβ groups was 146.1%,130.7% and 110.5%,respectively.

CONCLUSIONSThe results suggest that Livin isoform,especially Livinα+β is hopeful to be a molecular target for increasing sensitivity of lung cancer cell to chemotherapy.Gene silencing may be a new means of gene therapy for non-small cell lung cancer.

BACKGROUNDIt has been proven that mitochondrial DNA (mtDNA) noncoding region plays a very important role in process of transcription. The mutation in mtDNA noncoding region can influence the transcription and translation process of mtDNA. The aim of this study is to investigate the variation status in noncoding region of mtDNA in lung cancer cell lines, and discuss its meaning in carcinogenesis.

METHODSThe 7 lung cancer cell lines, A549, SPC-A-1, Calu-3, LTEP-a-2, QG-56, 95-D and NCI-H446, were cultured. Then the mtDNA of them was extracted with one-step method and variation status of noncoding region was analyzed, compared to the Cambridge sequence of mtDNA.

RESULTSDifferent variations existed in the mtDNA noncoding regions of 7 lung cancer cell lines. The variation rate of LTEP-a-2 (1.82%) was the highest one, and A549 (0.21%) was the lowest one.

CONCLUSIONSThe mtDNA noncoding region sequencing is accomplished firstly in 7 lung cancer cell lines. It may provide a reference and background for related researches.

BACKGROUNDMitochondrial DNA (mtDNA) is the only hereditary substance besides nucleus, which is composed of a code region and a non-code D-loop region. The aim of this study is to investigate the hypervariable region II (HVRII) mutation of mtDNA in peripheral blood leucocyte, pericancerous tissues and cancer tissues of lung squamous cell carcinoma patients, and to explore its significance.

METHODSWhite blood cells, pericancerous tissues and cancer tissues were obtained from 15 cases of lung squamous cell carcinoma patients and mtDNA were extracted by one step method. HVRII fragments were amplified by PCR. Mutations were determined by DNA sequencing and the mutations of HVRII were analysed.

RESULTSIn 15 lung squamous cell carcinoma patients, 14 patients showed mutation in HVRII(93.33%), 88 mutations were found totally. Eighty-seven mutations located in H-strand origin region, especially in the conserved sequence blocks and the mtTF1, 2 binding site (TFX and TFY).

CONCLUSIONSThe results suggest that the mutation frequency of HVRII in cancer tissues of lung squamous cell carcinoma patients is very high and it might play an important role in carcinogenesis of the lung.

BACKGROUNDp16INK4a and p14ARF, encoded by gene INK4a/ARF located at chromosome 9p21, are cyclin dependent kinase (CDK) inhibitors. Both p16INK4a and p14ARF are cell cycle regulatory proteins and play an important role in Rb and p53 passways respectively. In this study, wild-type INK4a/ARF gene was transfected into human lung adenocarcinoma cell line A549, in which this gene site was lost, and the effects on the cell's biological behavior were investigated.

METHODSThe recombinant eukaryotic expression plasmids pcDNA3-p16INK4a and pcDNA3-p14ARF were transfected into A549 by cationic liposome method. By RT-PCR, immunocytochemistry and Western blot after G418 selection, A549 cells that could stably express p16INK4a and p14ARF were obtained. As a control, the parental cell and negative control cell with plasmid pcDNA3-LacZ were used. Inhibition of proliferation was measured by MTT assay. The cell growth curve was drawn according to cell counts. Cell cycle distribution was measured by flow cytometry (FCM), the apoptosis indexes were observed at the same time. The colony formation rate was counted by staining the cells with Coomassie brilliant blue.

RESULTSThe introduction of exogenous INK4a and ARF caused significantly growth inhibition of A549. By FCM, more percentage of A549-p16INK4a-p14ARF cells couldn't pass through the checkpoint G1. The percentage of A549-p16INK4a-p14ARF cells inhibited at G0/G1 was 59.9%, 50.3% for A549-vector and 51.2% for A549. The statistical differences were significant between A549-p16INK4a-p14ARF cell and A549-vector cell (P=0.025) and between A549-p16INK4a-p14ARF cell and A549 cell (P=0.043). The apoptosis index of A549-p16INK4a-p14ARF cell was 8.0% and 2.7% for both A549-vector and A549 cell (P < 0.01). The colony formation ability of A549-p16INK4a-p14ARF was weaker than that of A549-vector and A549, they were 63%, 87% and 85% respectively.

CONCLUSIONSThe wild-type INK4a/ARF gene can be co-introduced effectively into A549 cell by cationic liposome method. The reexpression of p16INK4a and p14ARF in A549 can inhibit the growth and enhance the apoptosis. This trial will be helpful in using gene therapy of lung cancer in the future.

BACKGROUNDThe relationship between mitochondrial DNA (mtDNA) mutation and tumor is the hot point in cancer research field by now. The existent methods for extracting mtDNA are time-consuming and complicated. In order to further research the relationship of the mutation of mtDNA and development of lung cancer, it is necessary to establish a rapid and simple method for extracting mtDNA from lung cancer tissue.

METHODSLung cancer tissues were lysed in base-denaturalization liquid and mtDNA was directly extracted from them by chloroform:isoamyl alcohol (24:1). The mtDNA was confirmed by PCR.

RESULTSThe extracted mtDNA samples were not combinated with protein and 1528bp PCR products of (mtDNA) were detected from 40 samples of analyzing patients. About (7.97±0.12)μg of mtDNA could be extracted from 1 gram of cancer tissue.

CONCLUSIONSThe method is simple, rapid and efficient to extract (mtDNA) from human lung cancer tissues and it could be used to handle small amount tissues or large number of samples in clinical and scientific research.

Objective To observe the changes of retinal sensitive cellular ultrastructure of rats in critical period of visual development.Methods Ten normal healthy Wistar rats were chosen,eight of which were neonatal rats and two were mature rats.The eight neonatal rats were randomized into four groups(n=2 in each group) and respectively sacrificed on postborn day 0,15,20,25.The eyeballs of the neonatal rats and the mature rats were resected and the retinas were observed by electron microscope.Results The rat retinal sensitive cellular ultrastructure on postborn day 0 was immature and the cellular arrangement was not clear.The organelle of sensitive cell in critical period developed mature gradually and the arrangement of them was very clear.Conclusion The retinal sensitive cell of rats develops and matures gradually in critical period.

Objective To explore the silence of B-cell specific Moloney murine leukaemia virus insertion site 1(Bmi-1)by RNA interference on the proliferation of human leukemia cell line K562 and its mechanisms.Methods Small interfering RNA(siRNA)targeting Bmi-1 gene was designed and double-stranded siRNA was chemically synthesized.After double-stranded siRNA was transfected into K562 cells with Lipofectamine 2000,the proliferation of K562 cells was detected by MTT colorimetry,cell cycle was determined by flow cytometry,and the expression of Bmi-1 and P16 were analyzed by Western blotting.Results The siRNA targeting human Bmi-1 gene effectively prolonged the double time of K562 cells,increased the percentage of cells at G1 phase,and the expression of Bmi-1 was significantly down-regulated but the expression of P16 was up-regulated.Conclusion The siRNA targeting human Bmi-1 gene inhibits the proliferation of K562 cells,and up-regulates the expression of P16 in the cells.

Objective To investigate the relationship between hypervariable regionsⅠ(HVRⅠ) mutation of mitochondrial DNA (mtDNA) and lung squamous carcinoma and to explore its significance in carcinogenesis. Methods White blood cells and carcinoma tissues were obtained from 13 cases of lung squamous carcinoma patients and mtDNA were extracted by one step method. HVRⅠfragments were amplified by PCR. Mutations were determined by DNA sequencing. Results In 13 lung squamous carcinoma patients, 8 cases showed mutation in HVR Ⅰ, and 30 mutations were found in 25 different nucleotide sites, in which 17 were point mutations and 13 were insertions and deletions, including a 10-bp deletion in one patient. Conclusion These results suggest that the mutation rate of HVRⅠsequence in lung squamous carcinoma tissue was relatively high, and point mutation might play an important role in lung carcinogenesis.