Objective: To compare the mitochondrial content, the relative amount of mtDNA, and mitochondrial functions between the young and aging WI-38 cells, so as to investigate the correlation between mitochondrial and aging. Methods: Human embryonic lung diploid cell line WI-38 was cultured and its viability was assayed by MTT assay; the content of mitochondrial protein was determined using BCA-100 Protein Quantitative Analysis Kit after mitochondria were fractionated by differential centrifugation; mtDNA relative content was measured by a competitive polymerase chain reaction (PCR) method; mitochondrial membrane potential was measured by flow cytometry; and NADH oxidase activity was measured by spectrophotometry. Results: Compared with the young cells, the aging cells had a longer time to form a monolayer, an obviously decreased cell viability and mitochondrial membrane potential (by 50%), and a decreased NADH oxidase activity, with the maximal reaction speed declining from 66.73 nmol/(mg protein · min) to 36. 01 nmol/(mg protein · min). Mitochondrial content in the aging cells([0.78 ± 0.02] mg/ml) was higher than that in the young cells([0.56 ± 0.03] mg/ml). Using 18S rDNA of nuclear as an internal reference, the relative amount of mtDNA in the aging cells (1.557 ± 0.072) was found to be obviously higher than that in the young cells (1.292 ± 0.068). Conclusion: The increase of mitochondrial contents and mtDNA relative amounts in aging cells may be one of the compensatory mechanisms for decreased mitochondrial function, which may provide an evidence for studying the correlation between mitochondrial and aging.

Adult ; Carcinoma, Renal Cell ; secondary ; Female ; Humans ; Kidney Neoplasms ; pathology ; Magnetic Resonance Imaging ; Vaginal Neoplasms ; secondary

The establishment of laboratory medicine reference system is an important way for routine detection results to achieve traceability.The complete reference system consists of reference measurement procedures,reference materials and reference laboratories.The reference measurement procedure is usually characterized by a thorough study with a small measurement uncertainty.Based on the advantages of high specificity and high sensitivity in quantitative analysis, mass spectrometry plays an important role in the research of the reference measurement procedures in laboratory medicine.In the past 40 years, mass spectrometry technology has been more and more widely applied in medicine, electrolytes, metabolites and substrates, non-electrolyte metals, non-peptide hormones, protein and vitamin.It provides a powerful guarantee for the precise detection of these well-defined measureds.

Objective To explore acute toxicity of succimer on mice.Methods Twenty Kunming mice(10 males and 10 females) weighting approximately (21.2?2.3)g were acclimatized for 3 days prior to dosing,then were divided into control group and experiment group with 10 mice in each group according to body weight.Fasted for 12 hours,the mice in experiment group received intragastric administration of 160mg DMSA in deionized water in 24 hours,and the control group received the same volume of deionized water,and then they were observed for 7 days.Blood was collected into heparinized-tubes by removal of eyeball.All mice were sacrificed and brain,heart,liver and kidney were removed and washed with normal saline.The activity or amount of BUN,Scr,AST,ALT,SOD, GSH-PX and MDA were analyzed.Results (1)Given 160rag DMSA in 24 hours,gastrointestinal symptoms were main side effects.During the observation,experiment group lost weight due to the decrease of food-intake ,and some mice had slight hydroabdomen.(2)High dose of DMSA caused a significant inhibition of GSH-PX(P0.05).The hepatic cell was damaged accord- ing to the significant raise of MDA in liver(P0.05),which was related to acute toxicity on liver.Conclusion Succimer could inhibit the antioxidarrt systems and could do damage to liver and kidney.

OBJECTIVETo observe the effect of drug-containing serum of Chinese traditional herbal decoction Weikangning (WKN) on growth of gastric cancer cell, expression of vascular endothelial growth factor (VEGF) and its receptors including KDR and Flt-1.

METHODSA total of 120 male Wistar rats were given high, medium and low-dose WKN. After the drug-containing serum was prepared, the gastric cancer cells MGC-803 of different dose groups were cultured with the drug-containing serum, respectively. The gastric cell growth was observed by using light microscope and flow cytometer,the expression of VEGF and its receptor Flt-1 was detected with SABC immunohistochemistry method and the mRNA expression levels of VEGF and its receptors including KDR and Flt-1 of different groups were detected with reverse transcription-polymerase chain reaction (RT-PCR), respectively.

RESULTSThe gastric cancer cell growth and cell cycle of the three medicated groups were significantly improved as compared with those of the control group (P <0. 01) ,the proportion of cells in G0 - G1 phase was increased,while the cells in S phase were decreased. It was shown that the apoptotic rates were increased in the medicated groups in a dose-dependent manner. The gray scales ( microm(2) ) of VEGF and Flt-1 in high, medium and low dose groups was 182. 44 +/-0. 54,178. 65 +/-0. 56,174. 80 +/-0. 81 and 168. 51 +/- 0. 81,162. 01 +/-0. 52,148. 20 +/-0. 69, respectively vs 147.82 +/-0. 15(P <0.01) and 144.31 +/-0.71 (P <0.01) in the control group. The mRNA expression of VEGF and its receptors significantly decreased in gastric cancer cells after cultured with WKN contained serum (P < 0. 01 ).

CONCLUSIONWKN has inhibitory effects on gastric cancer cell growth and mRNA expression of VEGF as well as its receptors KDR and Flt-1.

Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; therapeutic use ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Male ; Rats ; Rats, Wistar ; Stomach Neoplasms ; drug therapy ; enzymology ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism ; Vascular Endothelial Growth Factor Receptor-1 ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism

Adolescent ; Child ; Child, Preschool ; Female ; Hematologic Neoplasms ; complications ; Humans ; Infant ; Lung Diseases, Fungal ; drug therapy ; etiology ; Male

OBJECTIVETo characterize the genetic aberrations in pediatric acute lymphoblastic leukemia (ALL).

METHODSNinety ALL cases were enrolled in the study from January 2009 to November 2011. Chromosome banding analysis and fluorescence in situ hybridization (FISH) were used to detect genetic aberrations.

RESULTS(1) Chromosome analysis: 35 (53.0%) of 66 cases who had metaphase were abnormal, and 24 cases had no metaphase. (2) FISH analysis: among the 31 cases who had normal karyotypes and 24 who had no metaphase detected by chromosome banding technique, 7 (22.6%) and 14 (58.3%) cases were abnormal detected by FISH, respectively. There were no statistically significant differences compared with chromosome analysis (P = 0.655). Among these 55 ALL cases TEL/AML1, bcr-abl and MLL fusion genes were observed in 16 (29.1%), 3(5.5%) and 2(3.6%) cases, respectively. (3) Cytogenetic aberration was observed in 56 of total 90 ALL cases (62.2%).

CONCLUSIONSCytogenetic changes are common in childhood ALL. Conventional cytogenetic study could reliably detected chromosomal abnormalities for ALL with assessable metaphase. FISH should be used as a complementary method for ALL patients who have poor chromosomal morphology or no metaphase cells, and combination of both methods can improve the detection rate of genetic abnormalities in childhood leukemia.

Adolescent ; Child ; Child, Preschool ; Chromosome Aberrations ; Female ; Fusion Proteins, bcr-abl ; genetics ; Humans ; In Situ Hybridization, Fluorescence ; Infant ; Karyotyping ; Male ; Myeloid-Lymphoid Leukemia Protein ; genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics

OBJECTIVECord blood (CB) provides a rich source of stem cells for transplantation. CB transplantation has been used widely after myeloablative therapy. One major disadvantage of CB transplantation is delayed platelet engraftment. The aim of this study was to hasten platelet engraftment by investigating the ability of different hematopoietic growth factor combinations to generate large numbers of megakaryocyte (Mk) from CB and by evaluating the biologic characteristics and function of the expanded Mk.

METHODSCB samples were obtained at the end of normal full-term deliveries with informed consent. Mononuclear cells (MNCs) were isolated from CB using Ficoll density centrifugation. MNC population was positively selected for CD(34) expression by magnetic cell sorting (MACS). CD(34)(+) cells were cultured in serum-free and stroma-free medium containing the following two different cytokine combinations: thrombopoietin (TPO) + stem cell factor (SCF) + interleukin (IL) -3 + IL-6 and TPO + SCF. Cultures were characterized after 3, 7, 10 and 14 days by flow cytometry, colony forming unit-megakaryocyte (CFU-Mk) and maturation evaluation (Mk ploidy). The expanded Mk function was examined by the platelet activation in vitro and severe combined immunodiffiency (SCID) mice transplantation in vivo.

RESULTSDifferent results were observed with different culture conditions. With the first cytokine combination optimal expansion of CD(41)(+) cells was observed on day 10, but the optimal expansion of Mk progenitors (CD(34)(+)CD(41)(+)) was observed on day 7, with a median 121 and 44-fold increase at the starting cell dose. This result was also proven by CFU-Mk. The largest numbers of CFU-Mk were also observed on day 7. The degree of maturation of Mk cells also increased as suggested by DNA content of CD(41)(+) cells, which means that CD(34)(+) cells cultured for 3 - 7 days were richer in primitive Mks, while those cultured for 10 - 14 days had greater numbers of more differentiated Mks. For the second cytokine combination, CD(41)(+) and CD(34)(+)CD(41)(+) cells were fewer than the first one, but it produced 36 and 85-fold CD(34)(+)CD(41)(+) and CD(41)(+) respectively on day 7. Platelet activation test confirmed that the expanded Mks had normal function. Therefore, the expanded Mks could be transplanted into the SCID mice bone marrow and produce human platelet in the peripheral blood of the mice.

CONCLUSIONEx vivo expanded Mk might facilitate CB transplantation and help shorten the period of post-transplant thrombocytopenia.

Animals ; Antigens, CD34 ; Cell Culture Techniques ; methods ; Cells, Cultured ; Culture Media ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Megakaryocytes ; cytology ; Mice ; Mice, SCID

BACKGROUND:Studies have shown that bioactive molecules or polypeptides grafted onto the surface of polyethylene glycol (PEG) hydrogels can improve PEG bioactivities.OBJECTIVE:To manufacture PEG hydrogels capable of auto-recruiting growth factor beta1 (TGF-β1) and to study its biocompatibility. METHODS:Pure PEG hydrogels (group A), PEG hydrogels grafted on a cell adhesion peptide RGD peptides (group B), PEG hydrogels grafted with auto-recruited TGF-β1 peptide sensitive polypeptide HSNGLPL (group C), PEG hydrogels grafted with both RGD and HSNGLPL polypeptides (group D) were prepared. Contract angle of the hydrogel was detected in each group. Human bone mesenchymal stem cells were seeded onto four kinds of hydrogels. After cells attached, scanning electron microscope and LIVE/DEAD staining were done to observe cell-hydrogel compounds. Human bone marrow mesenchymal stem cells were co-cultured with ordinary culture medium (control) or four kinds of hydrogels for 1, 3, 5, 7 days, and the cell proliferation was detected by cell counting kit-8 assay. The four kinds of hydrogels were put into 24-well culture plates with addition of PBS containing TGF-β1, and 1 hour later, immunofluorescence staining was done. RESULTS AND CONCLUSION:(1) The contact angles of groups A and C were larger than those of groups B and D. (2) Under the scanning electron microscope, groups A and C had little cells attached on the hydrogel surface, but there were many cells on the hydrogel surface in groups B and D. (3) LIVE/DEAD staining showed groups A and C had little living cells, and conversely groups B and D had many living cells. (4) The results of cell counting kit-8 demonstrated that as the incubation time went on, cell proliferation activity of five different groups increased with no difference at the same time point. (5) Findings from the immunofluorescence staining showed that groups A and B had very weak fluorescence, while groups C and D had stronger green fluorescence. In conclusion, PEG hydrogels grafted with RGD and HSNGLPL polypeptides can auto-recruit TGF-β1, and have good biocompatibility.

Objective To investigate the digestion kinetics of Apolipoprotein A-I and B by ID-LC-MS method for accurate quantification of proteins .Methods Methodological research .The target peptides of ApoA-I and B were determined .The ApoA-I and B from 5 human serum samples on market with levels from 0.90-2.54 g/L and 0.54-1.39 g/L separately , were measured in terms of target peptides by isotope dilution liquid chromatography mass spectrometry method .The releasing amount and rate of peptides were analyzed and plotted according to different time points .The correlation coefficient R2 was calculated among peptide releasing amount between samples .Results Most peptides reached their peaks within 4 hours.The peptides VQ , DY and VS from Apo A-I, TR and FP from Apo B were released relatively slowly .After getting to their peak stage , the ratio between TEV and SIL-TEV, AK and SIL-AK, VQ and SIL-VQ presented stable state.As for Apo A-I the correlations among peptides are high , from 0.904 to 0.999.Some peptides from Apo B show lower correlations , such as TG-SV with R20.543 (3 h).Conclusions Peptides from Apo A-I and Apo B present different releasing properties after trypsin digestion .Proper selection of representative peptides and enzymatic conditions can benefit accurate quantification of target proteins .