Factor analysis of effective platelet-producing ability of fetal liver-derived cells / 中华内科杂志

Objective:To study the different factors affecting platelet production post transplantation of hematopoietic stem cells (HSCs) isolated from different sources in order to explore novel options for treating platelet depletion following HSCs transplantation.Methods:HSCs and their downstream derivatives including myeloid and lymphoid cells (i.e., collective of mononuclear cells (MNCs)), were isolated from E14.5 fetal liver (FL) and bone marrow (BM) of 8-week-old mice by Ficoll separation technique. These cells were subsequently transplanted into the tibia bone marrow cavity of recipient mice post lethal myeloablative treatment in order to construct the FL-MNCs and BM-MNCs transplantation mouse model. Routine blood indices were examined in these recipient mice. The chimeric rate of donor cells in recipient peripheral blood cells were determined by flow cytometry. Different groups of cells involved in platelet reconstruction were analyzed. CD41 +megakaryocytes were sorted from fetal liver or bone marrow using magnetic beads, which were then induced to differentiate into platelets in an in vitro assay . Quantitative RT-PCR was used to detect the expression of platelet-related genes in CD41 +megakaryocytes from the two sources. Results:Both the FL-MNCs and the BM-MNCs transplantation groups resumed normal hematopoiesis at the 4th week after transplantation, and the blood cells of the recipient mice were largely replaced by the donor cells. Compared with the mice transplanted with BM-MNCs, the platelet level of mice transplanted with FL-MNCs recovered faster and were maintained at a higher level. At week 4, the PLT level of the FL-MNCs group was (1.45±0.37)×10 12/L, and of the BM-MNCs group was (1.22±0.24)×10 12/L, P<0.05. The FL-MNCs contain a higher proportion of hematopoietic stem cells (Lin -Sca-1 +c-Kit +)(7.60%±1.40%) compared to the BM-MNCs (1.10%±0.46%), P<0.01; the proportion of the megakaryocyte progenitor cells (Lin -Sca-1 -c-Kit +CD41 +CD150 +) and mature megakaryocyte cells (CD41 +CD42b +), also differ significantly between the FL-MNCs (3.05%±0.22%, 1.60%±0.06%, respectively) and the BM-MNCs (0.15%±0.02%, 0.87%±0.11%, respectively) groups, both P<0.01. In vitro functional studies showed that FL-MNCs-CD41 +megakaryocytes could produce proplatelet-like cells more quickly after induction, with proplatelet-like cells formation on day 3 and significant platelet-like particle formation on day 5, in contrast to bone marrow-derived BM-MNCs-CD41 +megakaryocytes that failed to form proplatelet-like cell on day 5. In addition, FL-MNCs-CD41 +cells expressed higher levels of platelet-related genes, Mpl (3.25-fold), Fog1 (3-fold), and Gata1 (1.5-fold) ( P<0.05). Conclusion:Compared with the BM-MNCs group, the FL-MNCs transplantation group appears to have a more efficient platelet implantation effect in the HSCs transplantation recipient in vivo , as well as a higher platelet differentiation rate in vitro. This might be related to a higher proportion of megakaryocytes and higher expression levels of genes such as Mpl, Fog1, and Gata1 that could be important for platelet formation in FL-MNCs-CD41 +cells. Further exploration of the specific functions of these genes and the characteristics of the different proportions of the donor cells will provide valuable clues for the future treatment of platelets reconstitution after HSCs transplantation clinically.

Status and advances of long-acting factor Ⅷ / 生物工程学报

Current treatment for hemophilia A is based on replacement therapy that is the most effective method by using recombinant clotting factor FⅧ (rFⅧ). Although the safety and effectiveness of replacement therapy has been proved by clinical practice for the last decades, FⅧ products are temporally limited because of a short half-life and requiring prophylactic injections frequently for most patients, usually three times per week or every other day. Frequent intravenous injection not only brings physical pain to the patient, but also produces FⅧ antibodies that seriously affect the treatment effect. In this paper, we review the present status, research progress and main problems of the long-acting recombinant factor Ⅷ.

The effect of DNA hydroxymethylase Tet2 on γ globin activation in the treatment of β-thalassemia / 中华内科杂志

Objective To study the function of ten-eleven translocation 2 (Tet2) in γglobin gene expression in patients with β-thalassemia.Methods Gamma globin expression was induced by 5-azacytidine and Tet2 gene expression was knocked down by short hairpin RNA (shRNA) in a human immortalized myelogenous leukemia K562 cell line.The global 5-hydroxymethylcytosine (5hmC) level was measured by an ELISA kit.5hmC level of γglobin gene was quantified by sulfite sequencing.The mRNA level of Tet2,γglobin,and related transcription factors Nfe4 and Klfl were quantified by real-time PCR.Results Tet2 knockdown resulted in a decreased global 5hmC level from 0.14％ to 0.03％ as of the control group in K562 cells.The expression of γ globin was enhanced after 5-azacytidine treatment in vitro.However,γglobin mRNA level in Tet2 knockdown cells was only 55％ as that in control group.The CG sites on γ globin gene were unmethylated.As Tet2 was down-regulated,the expression levels of Nfe4 and Klf1 decreased by about 80％ and increased to 3.5 folds,respectively.Conclusions Tet2 appears to maintain 5hmC level and facilitates γ globin gene activation.Moreover,Tet2 more likely regulates γglobin expression via affecting transcription factors rather than the gene itself.Thus,Tet2 could be a potential therapeutic target for β thalassemias.

Hemophilia B replacement therapy drugs / 生物工程学报

Hemophilia B is an X chromosome linked hereditary hemorrhagic disease, which is caused by the lose function mutation of factor IX (FIX), and significantly affects the patients' lifespan and life quality. The severity of hemophilia B depends on the FIX level in the plasma. By referring to the relevant literatures, we reviewed and summarized hemophilia B replacement therapies. Specifically, we focus on recombinant factor IX products on the market and those in the pipeline, especially on the long-acting factor IX drugs, to provide the basis for researches of new hemophilia B drugs.

Prevention of pancreatitis after endoscopic retrograde cholangiopancreatography with different methods:a Meta analysis / 中华消化外科杂志

Objective To evaluate the efficacy of different methods in preventing pancreatitis after endoscopic retrograde cholangiopancreatography (ERCP).Methods Databases including PubMed,EMBASE,Cochrane Library,Chinese Journal Full-text Database,China Biomedicine Database were searched with key words including endoscopic retrograde cholangiopancreatography,ERCP,post-ERCP pancreatitis,pancreatitis,pancreatic duct stent,non-steroid anti-inflammatory drugs,indometacin,diclofenac,protease inhibitors,nafamostat,ulinastatin,gabexate,somatostain,内镜逆行胰胆管造影,内镜逆行胰胆管造影术后胰腺炎,胰腺炎,胰管支架置入,非甾体类抗炎药,吲哚美辛,双氯芬酸,抑酶剂,萘莫司他,乌司他丁,加贝酯and生长抑素.Literatures published between January 2000 and January 2014 were searched.Randomized controlled studies on prevention of pancreatitis after ERCP which were enrolled in this study were analyzed by 2 independent reviewers.The quality of the literatures was evaluated.All data were analyzed using the RevMan 5.0 software.Data were expressed in odds ratio (OR) and 95％ confidence interval (95％ CI).The heterogeneity of the studies was analyzed using the I2 test.Results Twenty-seven literatures were enrolled in the study.There were 4 701 patients in the experimental group (including patients who were treated by pancreatic stent installation,non-steroidal antiinflammatory drugs,nafamostat,ulinastatin,gabexate,intravenous infusion of somatostain for more than 6 hours,intravenous infusion of somatostain for less than 6 hours,bolus injection of somatostain) and 3 592 patients in the control group (including patients treated without pancreatic duct installation or placebo).The results of Meta analysis showed that pancreatic stent installation,non-steroid anti-inflammatory drugs,nafamostat,intravenous infusion of somatostain for more than 6 hours and bolus injection of somatostain could significantly decrease the incidence of pancreatitis after ERCP (OR =0.18,0.45,0.31,0.33,0.25,95％ CI:0.09-0.35,0.33-0.61,0.19-0.52,0.20-0.56,0.11-0.55,P ＜ 0.05).Conclusion Pancreatic stent installation,non-steroid anti-inflammatory drugs,nafamostat,intravenous infusion of somatostain for more than 6 hours and bolus injection of somatostain could effectively prevent the incidence of pancreatitis after ERCP.

Isolation and gene modification of amniotic fluid derived progenitor cells / 生物工程学报

We established methods to isolate human amniotic fluid-derived progenitor cells (hAFPCs), and analyze the ability of hAFPCs to secrete human coagulation factor IX (hFIX) after gene modification. The hAFPCs were manually isolated by selection for attachment to gelatin coated culture dish. hFIX cDNA was transfected into hAPFCs by using a lentiviral vector. The hFIX protein concentration and activity produced from hAFPCs were determined by enzyme-linked immunosorbent assay (ELISA) and clotting assay. The isolated spindle-shaped cells showed fibroblastoid morphology after three culture passages. The doubling time in culture was 39.05 hours. Immunocytochemistry staining of the fibroblast-like cells from amniotic fluid detected expression of stem cell markers such as SSEA4 and TRA1-60. Quantitative PCR analysis demonstrated the expression of NANOG, OCT4 and SOX2 mRNAs. Transfected hAFPCs could produce and secrete hFIX into the culture medium. The observed concentration of secreted hFIX was 20.37% +/- 2.77% two days after passage, with clotting activity of 16.42% +/- 1.78%. The amount of hFIX:Ag reached a plateau of 50.35% +/- 5.42%, with clotting activity 45.34% +/- 4.67%. In conclusion, this study established method to isolate and culture amniotic fluid progenitor cells. Transfected hAFPCs can produce hFIX at stable levels in vitro, and clotting activity increases with higher hFIX concentration. Genetically engineered hAFPC are a potential method for prenatal treatment of hemophilia B.

Directed hepatic differentiation from embryonic stem cells

The liver is the largest internal organ in mammals, and is important for the maintenance of normal physiological functions of other tissues and organs. Hepatitis, cirrhosis, liver cancer and other chronic liver diseases are serious threats to human health, and these problems are compounded by a scarcity of liver donors for transplantation therapies. Directed differentiation of embryonic stem cells to liver cells is a promising strategy for obtaining hepatocytes that can be used for cell transplantation. In vitro hepatocyte differentiation of embryonic stem cells requires a profound understanding of normal development during embryonic hepatogenesis. Here we provide a simple description of hepatogenesis in vivo and discuss directed differentiation of embryonic stem cells into hepatocytes in vitro.

Establishment and application of flow cytometry in detecting human cells in human/goat chimera models / 中华检验医学杂志

Objective To establish the methodology of flow cytometry for detecting human cells in human/goat chimerisra.Methods Human hemopoietic stem/progenitor cells (CD+34 cells) or MIG-tranadueed-GFP CD+34 cells were transplanted into the peritoneal cavity of fetal goats in utero to obtain human/goat chimera modeL The peripheral blood cells from the chimeras were labeled with multiple mouse anti-human antibodies and the monoelonal antibodies that were specific for human but had not or only minimal cross-reaction with goat were screened as the primary antibodies for routine analysis in flow cytometry.Human cord blood was proportionally (25% ,50% ,75%,100%) added into the blood of the untransplanted goats and the cells were labeled with CD+34 monoclonal antibody.The region and size of the "gate" were chosen based on to the distribution of CD+34 cells or human cord blood.One human/goat chimera marked with GFP (MIG goat) was sacrificed and the substantial liver cells from its perfused liver were analyzed for the GFP+cells percentage and DNA contents by flow cytometry.Results CD7,CD15,CD38,CD45CD20CD34CD14and GPA monoclonal antibodies were chosen as the primary antibodies in rou tine detection by flow cytometry.The size and area of the "gate" were also defined.29.1% (29100/100 000 ) of the substantial liver cells from the MIG goat expressed GFP.DNA content analysis showed that the GFP+ cells obtained from the liver of MIG goat mainly manifested two peaks that were correspond to those of human.Conclusions Flow cytometry is rapid,simple and effective for the investigation of differentiation,homing and biological characteristics of stem cells in vivo.The selections of suitable surface antibodies and the "gate" are very important for detecting human cells accurately in the human/goat chimerism.

An Expedient Reliable Double Fluorescent Reporter System for φC31 Integrase Function Evaluation / 生物化学与生物物理进展

A reporter system for φC31 integrase was developed in NIH3T3 cells. The reporter plasmid coding green fluorescent protein (GFP) coupled with red fluorescent protein (RFP) was eo-transfected with the plasmid coding φC31 integrase, to show the activity of integrase in the cells. Fluorescence activated cell sorter (FACS) was used to measure the proportion of the cells containing red and green fluorescence. The increment of green cells was positively related to the increase in the transfection with plasmid coding φC31 integrase. Approximately 90% of green cells were observed under a ratio of [plasmid-φC31-integrase]/[reporter plasmid] at 10 : 1. This suggests that the φC31 integrase reporter system provides a probe for the function of φC31 integrase in cells.

Regulation on the expression of bovine prolactin gene by different promoters / 生物工程学报

To compare the regulation effects by different promoters on bovine prolactin gene expression in different cell lines, three recombinant bovine prolactin expression vectors were constructed using different promoters, i.e., CMV promoter, bovine prolactin gene promoter and goat beta-casein gene promoter, respectively named pCMV, pPRLP and pP1A3, which were transfected into two cell lines, mouse pituitary tumor cell strain (AtT20) and mouse mammary epithelial cell strain (HC11), respectively. RT-PCR and real-time RT-PCR were used to investigate the expression level of the above three vectors in both cell lines, pCMV vector was effectively expressed in both cell lines, pPRLP vector had a similar expression level to that of pCMV in both cell lines, pP1A3 was expressed in HC11 but not in AtT20. pP1A3 was tissue-specific to mammary gland, pPRLP was able to express with a significant level in pituitary and mammary glands, while its tissue-specific characteristics in other tissues need further investigation.

An Expedient Reliable Double Fluorescent Reporter System for ?C31 Integrase Function Evaluation / 生物化学与生物物理进展

A reporter system for ?C31 integrase was developed in NIH3T3 cells.The reporter plasmid coding green fluorescent protein(GFP) coupled with red fluorescent protein(RFP) was co-transfected with the plasmid coding ?C31 integrase, to show the activity of integrase in the cells.Fluorescence activated cell sorter(FACS) was used to measure the proportion of the cells containing red and green fluorescence.The increment of green cells was positively related to the increase in the transfection with plasmid coding ?C31 integrase.Approximately 90% of green cells were observed under a ratio of plasmid-?C31-integrase/reporter plasmid at 10∶1.This suggests that the ?C31 integrase reporter system provides a probe for the function of ?C31 integrase in cells.