ObjectiveTo summarize the current research progress of serum exosome microRNAs in patients with colorectal cancer.MethodsThe domestic and foreign literatures related to serum exosome microRNAs of colorectal cancer patients, which had been reported in recent years were collected through literature search. Subsequently, those literatures were used to read and review.ResultsExosomes were extracellular vesicles, which contained lipids, proteins, DNA, RNA (mRNA, microRNA, and long non-coding RNA), and other molecules. These vesicles mediated communication between cells by transporting the above molecules. Exosomes in serum were the main carriers of microRNAs in the blood circulation system. Serum exosome microRNAs could affect the proliferation, invasion, and metastasis of colorectal cancer cells, mediate the drug resistance of colorectal cancer cells, and could be used as biomarkers to predict the prognosis of colorectal cancer.ConclusionsSerum exosome microRNAs play important role in the occurrence, development, treatment, and diagnosis of colorectal cancer. As a class of biomarker, serum exosome microRNAs have great potential in the early diagnosis and prognostic evaluation of colorectal cancer.
Abstract： Objective To study the molecular mechanism of pathologic states related to cardiopulmonary bypass (CPB) and screen the differential proteins from the serum before and after CPB in the open heart surgery patients. Methods By the twodimensional gel electrophoresis (2DE), we took the blood samples from each of the sixteen open heart surgery patients 30 minutes before CPB, 1 hour after CPB, and 24 hours after CPB. The protein spots were analyzed by the PDQuest image analysis software and the differential protein spots were identified by matrixassisted laser desorption/ionizationtime of flightmass spectrometry (MALDITOF-MS). Then, enzymelinked immunosorbent assay (ELISA) was used to determine the expression level of serum amyloid A protein (SAA) in the serum of healthy people and the enrolled patients before and after CPB. Results Through 2DE in combination with massspectrometry, 7 proteins altered in expression were identified, including SAA, haptoglobin (HPT), leucinerich alpha2-glycoprotein (A2GL), hemoglobin subunit beta (HBB), serine/threonineprotein phosphatase 2A -regulatory subunit B″ subunit gamma (P2R3C), transthyretin (TTHY), and T-complex protein 11-like protein2 (T11L2). ELISA analysis showed that SAA levels in healthy people and the open heart surgery patients 30 minutes before CPB were not statistically different (t=-1.955, P=0.056), while the SAA level rose from 54.47±48.32 μg/ml 30 min before CPB to 1 017.78±189.92 μg/ml 24 hours after CPB in the serum of open heart surgery patients． Conclusion The results of this pilot study illustrate that SAA, HPT, A2GL, HBB, P2R3C, TTHY and T11L2 may be the molecule markers of pathologic state related to CPB. Acute phase reaction happens intensively after CPB in human body.
Delirium is an acute cognitive disorder caused by a variety of factors which lead to cerebral cortical dysfunction. At present the studies on the pathophysiology of delirium is still very few. But studies on serum biomarker of delirium can help to elucidate the pathophysiological mechanism of delirium, and the studies are significant for delirium diagnosis, severity classification and prediction of long-term outcome. This review examines three major groups of delirium related serum biomarkers: ① risk markers: those that are present or elevated prior to disease onset, including serum chemistries, genetic markers and so on; ② disease markers: those markers elevate with delirium onset and fall when delirium recovery, including acetylcholine and serum anticholinergic activity, serotonin, serum amino acids, and melatonin, interleukin, C-reactive protein; and ③ end products: those that rise in proportion to the consequences of disease, including S-100? and neuron specific enolase. The three markers mentioned above are helpful to further investigate the mechanism of delirium.
ObjectiveTo investigate the most appropriate culture time with the action of EGF in colon cancer stem cells enrichment by suspension culture.MethodsDLD-1 cells were cultured in serum-free medium containing 20 ng/mL EGF to generate spheroid cells. The time gradient was set to 10 d, 20 d, 30 d and 40 d, the cell proportion of CD133+, CD44+ and CD133+CD44+ were confirmed by flow cytometery. The ability of self-renewal was detected by the sphere forming assay and the limited dilution assay, and the in vitro tumorigenicity of the cells was detected by the colony formation assay.ResultsIn the 30 d group, the proportion of CD133+ and CD133+ CD44+ cells were significantly higher than those in the other groups (allP<0.05), the CD44+ cell was higher than that in the 20 d group (P<0.05), but there was no significant difference with the other two groups (P>0.05). The results of the limited dilution assay and the colony formation assay, the number of spheres in the 30 d or 40 d group was the highest among the 4 groups, and there was no statistical difference between the 30 d group and 40 d group (P>0.05), with statistically significant difference between the 30 d, 10 d and 20 d groups (all P<0.05). The results of the sphere forming assay and the self-renewal ability of 30 d group was significantly higher compared with other groups (all P< 0.05).ConclusionThe cancer stem cells could be enriched more efficiently by suspension culture using 20 ng/mL EGF for 30 days.
The nucleic acid adapters of tumor serum markers are oligonucleotide molecules with high specificity and high affinity with tumor serum markers obtained by in vitro screening with systematic evolution of ligands by exponential enrichment (SELEX). Researchers take the advantage of the nucleic acid adapter to explore new tumor serum markers that have more diagnostic value for tumor diagnosis. Recently, some achievements have been achieved in the research of liver cancer and stomach cancer. This paper has reviewed nucleic acid adapter and its research in the serum tumor marker screening, and discussed the value of the nucleic acid adapter of serum tumor markers in the diagnosis, as well as current problems existing in the research. This paper is very useful to help people better understand the screening of nucleic acid adapters of tumor serum markers, and to provide help in discovering new tumor serum markers.
Hypertensive disorder of pregnancy (HDP) is a type of disease unique to women during pregnancy. The most common clinical types are gestational hypertension and preeclampsia, which seriously threaten the health of pregnant women and fetuses. At present, there are no established criteria for the prediction and prevention of HDP. In recent years, a large number of studies have been carried out on HDP around the world, and many studies have shown a close correlation between serum uric acid and HDP. This article reviews the results of existing literature, elucidates the relationship between serum uric acid and the pathogenesis of HDP, prediction of HDP occurrence and development, and adverse pregnancy outcomes.
Objective To investigate the effect of ultra-filtration on reducing the matrix effects of the immersionof recombination human acellular dermal matrix (rhADM) on detecting residual bovine serum albumin (BSA) by ELISA.Methods Preparation of rhADM immersion: rhADM were rinsed, and then rhADM immersion were prepared. Physiologicalsal ine was used as immersion medium. Presaturation and ultra-filtration: marked the ultra-filtration tubes as PR1 (presaturation protocol 1), PR2 (presaturation protocol 2) and rhADM, respectively, added 2 mL of 1 mg/mL and 10 μg/mL BSA solution into PR1 and PR2 respectively, and added 2 mL of rhADM immersion into rhADM tubes (rhADM1 and rhADM2). The tubes were then centrifuged at 1 500 × g for 20 minutes. The above steps were repeated for 3 times. Take the inner-tube of ultrafiltration into unused centrifuge tube. Added 4 mL of 10 μg/mL BSA solution in PR1 and PR2 tubes, 4 mL of rhADM immersion in rhADM tubes, centrifuged at 1 500 × g for 20 minutes, and then the filtration was colleted. Detecting BSA concentration: the BSA concentrations of all samples were detected by using the quantitative measure of residual BSA ELISA kit. The recoveries of 10 μg/ mL BSA solution treated by presaturation protocol 1 and 2 were calculated (untreated 10 μg/mL BSA solution was as the basic sample, marked R10 and R20 respectively). The correlation coefficient between the logarithm of the filtrate dilution and the absorbance (A) value was calculated and compared with that of water exact without ultra-filtration. Results The BSA concentration of PR1 and R10 was (23.80 ± 1.58) μg/ mL and (9.04 ± 0.24) μg/mL, respectively. The BSA concentration of PR2 and R20 was (8.64 ± 0.24) μg/mL and (8.12 ± 1.01) μg/ mL, respectively. The average recovery of 10 μg/mL BSA was 263.4% ± 16.9% and 106.5% ± 3.0% when the ultra-filtration tubes were presaturaed by PR1 and PR2 (P lt; 0.01), respectively. The BSA recovery of PR2 met the detecting demand. The correlations between A value and sample dilution were increased, the correlationcoefficient was raised from — 0.727 to — 0.960 after rhADM immersion were treated by ultra-filtration. Conclusion Theresults show that the matrix effects can be reduced effectively by ultra-filtration, indicating that an acceptable recovery of BSA can be acquired when ultra-filtration tube is presaturated by sample water extract.
Objective
The aim is to sort CD90+ subpopulation cells in human liver cancer cell lines and investigate efficiency of magnetic cell sorting (MACS) on sorting the liver cancer stem cells.
Methods
①Expressions of CD90. Immunohistochemical method was used to determine the expressions of CD90 in normal liver tissues in 8 cases, liver cancer and adjacent liver cancer tissues in 58 cases. ②Screened the cell lines. Huh-7, MHCC97-H, Bel-7402, and SMMC-7721 cell lines were divided into blank control group and experimental group (5.5×105 cells per hole, 1 hole), cells of the experimental group were added with 5 μL CD90–PE while cells of the blank control group were treated with 5 μL CD90–PE non fluorescent antibody. Determined the proportion of CD90+ cells in the 2 groups by flow cytometry (FCM). ③MACS. Huh-7 and MHCC97-H cell lines were labeled with magnetic beads respectively and sorted by MACS, 1 mL cell suspensionsorted by magnetic sorting (MS) was collected as CD90– group, and 1 mL PBS after MS wash was collected as CD90+ group, as well as blank control group and experimental group. Determined the proportion of CD90+ cells in 4 groups by FCM. Two times of MACS were performed in Huh-7 cells. ④Serum free culture and serum culture. Huh-7 cells were divided into serum-free culture group and serum culture group (1 hole), and proportions of CD90+ cells were determined by FCM at 1 week after culture.
Results
①The positive rate of CD90 was 0 (0/8), 65.5% (38/58), and 20.7% (12/58) in normal liver tissues, liver cancer tissues, and adjacent liver cancer tissues respectively, and the positive rate of CD90 was higher in liver cancer tissues than those of normal liver tissues (χ2=6.78, P<0.05) and adjacent liver cancer tissues (χ2=20.83, P<0.05). ②For Huh-7, MHCC97-H, SMMC-7721, and Bel7402 cell lines, the proportions of CD90+ cells in the experimental group was 0.851%, 1.090%, 2.710%, and 4.050% respectively, the proportions of CD90+ cells in the blank control group was 0.241%, 0.688%, 1.890%, and 2.080% respectively, so we chose Huh-7 and MHCC97-H cell lines to perform MACS. ③Results of MACS for Huh-7 cell line. For the first MACS, the proportions of CD90+ cells in the blank control group, experimental group, CD90– group, and CD90+ group was 0.241%, 0.851%, 0.574%, and 1.100% respectively. For the second MACS, the proportions of CD90+ cells in the blank control group, experimental group, CD90– group, and CD90+ group was 0.032%, 0.961%, 0.426%, and 9.700% respectively.
Conclusions
The normal liver tissues do not express the CD90, but the liver cancer tissues express CD90 highly. There is a few CD90+ cells in Huh-7 and MHCC97-H liver cancer cell lines. The MACS has a certain effect on improving the proportion of CD90+ cells in the cell lines. The serum-free suspension culture has no effect on enriching CD90+ cells.
Objective To investigate the possibility of culturing human oral keratinocyte using autologous serum in order to provide theoretical and technical foundation for clinical application of tissue engineering oral mucosa epithelium.Methods The human oral keratinocytes were cultured by the medium containing different concentrations of autologous serum(10%,20%,30%)and fetalbovine serum (10%), respectively. The growth conditions for the cell and the mucosa epithelium in the groups were observed, the cell growth curves were drawn, and the population doubling time (PDT) was counted. Results The results showed that the human oral keratinocyte could proliferate well in the medium containing autologous serum or fetal bovine serum. The differences in the 24hour clone rate and PDT were not significant. Both the area and the thickness of the cultured oral epithelium increased with the increase of the autologous serum concentration, and the difference between autologous serum and fetal bovine serum was significant, especially with the medium containing 20% autologous serum( P＜0.05) . The human nature of the cultured epithelium was demonstrated by the immunofluorescent mouse anti-HLA antigen. Conclusion The autologous serum can replace the fetal bovine serum to culture the oral keratinocyte well, and the cultured oral mucosa epithelium can be better differentiated in the autologous serum than in the fetal bovine serum.
Objective To solve the shortage of hepatocytes for l iver tissue engineering, to explore the possibil ity of prol iferation of rat bone marrow mesenchymal stem cells (BMSCs) and the feasibil ity of differentiation of BMSCs into hepatocyteswith a culture system containing cholestatic rat serum and hepatocyte growth factor (HGF) in vitro. Methods Myeloid cellsof femur and tibia were collected from the female healthy Wistar rats at the age of 6 weeks, the BMSCs were isolated, purified and identified. Normal and cholestatic rat serum were prepared from 40 healthy Wistar rats at the age of 12-14 weeks. The 3rd passage of BMSCs were harvested and added different cultures according to the following grouping: group A, DMEM plus 10%FBS; group B, hepatocyte growth medium (HGM) plus 5%FBS; group C, HGM plus 5% normal rat serum; group D, HGM plus 5% cholestatic rat serum; group E, HGM plus 5% cholestatic rat serum plus 25 μg/L HGF. The changes of cell morphology were observed, MTT assay was used to measure cell growth; the expression of alpha-fetoprotein (AFP) and cytokeratin 18 (CK18) were detected by immunocytochemistry; the glycogen deposit was examined by periodic acid-schiff (PAS) staining; and the urea content in culture supernatant was determined by glutamate dehydrogenase. Results Polygonal cells and binuclear cells were observed in groups D and E, while the shapes of cells in groups A, B, and C did not obviously change. The cell growth curve demonstrated that the speed of cells proliferation in group C was the fastest, the one in group B was the slowest; showing significant differences when compared with groups A, D, and E (P lt; 0.05). On the 7th day in groups D and E, the positive expressions of AFP and CK18 emerged, on the 14th day the positive expression of glycogen emerged. At the same period, the expression ratio was higherin group E than in group D (P lt; 0.05). The urea concentration increased gradually with induction time in groups D and E, the concentration was higher in group E than in group D (P lt; 0.05). No expressions of AFP, CK18, glycogen, and change of the urea concentration were observed in groups A, B, and C. Conclusion Normal rat serum can obviously promote the growth of BMSCs; cholestatic rat serum which promote the growth of BMSCs can induce to differentiate into hepatocyte; and a combination of cholestatic serum and HGF can increase the differentiation ratio.
