AIM:To study the immunophenotype of hematopoietic progenitor cells from cord blood (CB) grafts (n = 39) in comparison with adult apheresis grafts (AG, n = 229) and pre-apheresis peripheral blood (PAPB) samples (n = 90...AIM:To study the immunophenotype of hematopoietic progenitor cells from cord blood (CB) grafts (n = 39) in comparison with adult apheresis grafts (AG, n = 229) and pre-apheresis peripheral blood (PAPB) samples (n = 908) using flow cytometry analysis.METHODS: First, we performed a qualitative analysis of CD34+ cell sub-populations in both CB and PAPB grafts using the standardized ISHAGE protocol and a wide panel of 20 monoclonal antibodies. Next, we stud-ied some parameters, such as the age of mothers and the weight of newborns, which can influence the qual-ity and the quantity of CD34+ cells from CB. RESULTS: We found that the percentage of apoptotic cells was high in CB in comparison to PAPB (PAPB: 4.6% ± 2.6% vs CB: 53.4% ± 5.2%, P < 0.001). In CB, the weight of newborn and the age of the mother have the influence on CD34+ cells. The follow-up of Ag CD133in the ISHAGE double platform protocol in association with CD45, CD34 and the 7’AAD shows an equal rate between the two cell populations CD133+CD45+CD34+ high and CD34+CD45+ high with a higher percentage. So, is the inclusion of Ac CD133 necessary in the pres-ent panel included in the ISHAGE methodflLast part, we showed a signif icant presence of interferon γ in CB in comparison to PAPB, the annexin showing the high number of apoptotic cells in CB. CONCLUSION: This study demonstrates that many different obstetric factors must be taken into account when processing and cryo-banking umbilical CB units for transplantation.展开更多
AIM:To investigate the effects of RNA interference tar-geting hepatocyte progenitor kinase-like kinase(HGK) in the invasion and adhesion of hepatocellular carcinoma(HCC) cell line HepG2.METHODS:Three paired insert DNA...AIM:To investigate the effects of RNA interference tar-geting hepatocyte progenitor kinase-like kinase(HGK) in the invasion and adhesion of hepatocellular carcinoma(HCC) cell line HepG2.METHODS:Three paired insert DNA fragments specif ic to HGK gene and one negative control DNA fragment were synthesized and inserted into RNAi-Ready pSIREN-RetroQ-ZsGreen vector.Western blotting assay and real-time reverse transcriptase polymerase chain reaction(RT-PCR) were used to screen the vector with a highest inhibitory rate.The vector was used to generate recom-binant retrovirus specif ic to HGK.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2h-tetrazolium bromide(MTT) assay was used to examine cell growth;wound closure assay and cell adhesion assay were employed to investigate cell migration and adhesion respectively;and transwell assay and three-dimensional culture invasion assay were used to detect cell invasion.The expressions of matrix metalloproteinase(MMP)-2,MMP-9 and nuclear factor(NF)-κB were detected by Western blotting assay.RESULTS:The real time RT-PCR and Western blotting assay showed that cells transfected with retrovirus me-diating RNAi targeting of HGK(RV-shHGK)-1 vector had the strongest inhibition of HGK protein,with an inhibi-tion rate of 76%,and this vector was used to generate recombinant retrovirus RV-shHGK-1.Cell adhesion assay and MTT assay found that cell adhesion and growth of the cells infected with RV-shHGK-1 were significantly lower than those of the control cells(P < 0.05).Wound closure assay,transwell assay and three-dimensional culture invasion assay showed that the cell invasiveness was significantly less in HGK knockdown cells than in the control cells(P < 0.05).The expressions of MMP-2,MMP-9 and NF-κB were inhibited in HepG2 cells infected with RV-shHGK-1.CONCLUSION:Down-regulation of HGK can obviously inhibit the migration and invasion of HepG2 cells in vitro.HGK may be a new therapeutic target for treatment of HCC.展开更多
文摘AIM:To study the immunophenotype of hematopoietic progenitor cells from cord blood (CB) grafts (n = 39) in comparison with adult apheresis grafts (AG, n = 229) and pre-apheresis peripheral blood (PAPB) samples (n = 908) using flow cytometry analysis.METHODS: First, we performed a qualitative analysis of CD34+ cell sub-populations in both CB and PAPB grafts using the standardized ISHAGE protocol and a wide panel of 20 monoclonal antibodies. Next, we stud-ied some parameters, such as the age of mothers and the weight of newborns, which can influence the qual-ity and the quantity of CD34+ cells from CB. RESULTS: We found that the percentage of apoptotic cells was high in CB in comparison to PAPB (PAPB: 4.6% ± 2.6% vs CB: 53.4% ± 5.2%, P < 0.001). In CB, the weight of newborn and the age of the mother have the influence on CD34+ cells. The follow-up of Ag CD133in the ISHAGE double platform protocol in association with CD45, CD34 and the 7’AAD shows an equal rate between the two cell populations CD133+CD45+CD34+ high and CD34+CD45+ high with a higher percentage. So, is the inclusion of Ac CD133 necessary in the pres-ent panel included in the ISHAGE methodflLast part, we showed a signif icant presence of interferon γ in CB in comparison to PAPB, the annexin showing the high number of apoptotic cells in CB. CONCLUSION: This study demonstrates that many different obstetric factors must be taken into account when processing and cryo-banking umbilical CB units for transplantation.
基金Supported by The Natural Science Foundation of China,No. 81071692
文摘AIM:To investigate the effects of RNA interference tar-geting hepatocyte progenitor kinase-like kinase(HGK) in the invasion and adhesion of hepatocellular carcinoma(HCC) cell line HepG2.METHODS:Three paired insert DNA fragments specif ic to HGK gene and one negative control DNA fragment were synthesized and inserted into RNAi-Ready pSIREN-RetroQ-ZsGreen vector.Western blotting assay and real-time reverse transcriptase polymerase chain reaction(RT-PCR) were used to screen the vector with a highest inhibitory rate.The vector was used to generate recom-binant retrovirus specif ic to HGK.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2h-tetrazolium bromide(MTT) assay was used to examine cell growth;wound closure assay and cell adhesion assay were employed to investigate cell migration and adhesion respectively;and transwell assay and three-dimensional culture invasion assay were used to detect cell invasion.The expressions of matrix metalloproteinase(MMP)-2,MMP-9 and nuclear factor(NF)-κB were detected by Western blotting assay.RESULTS:The real time RT-PCR and Western blotting assay showed that cells transfected with retrovirus me-diating RNAi targeting of HGK(RV-shHGK)-1 vector had the strongest inhibition of HGK protein,with an inhibi-tion rate of 76%,and this vector was used to generate recombinant retrovirus RV-shHGK-1.Cell adhesion assay and MTT assay found that cell adhesion and growth of the cells infected with RV-shHGK-1 were significantly lower than those of the control cells(P < 0.05).Wound closure assay,transwell assay and three-dimensional culture invasion assay showed that the cell invasiveness was significantly less in HGK knockdown cells than in the control cells(P < 0.05).The expressions of MMP-2,MMP-9 and NF-κB were inhibited in HepG2 cells infected with RV-shHGK-1.CONCLUSION:Down-regulation of HGK can obviously inhibit the migration and invasion of HepG2 cells in vitro.HGK may be a new therapeutic target for treatment of HCC.
