ObjectiveTo investigate the relationship between the expressions of B-cell-specific monoclonal leukemia virus insert site 1 (Bmi1) and human telomerase reverse transcriptase (hTERT) genes and the proliferative capacity of stem cells. MethodsCord blood CD34+ cells were cultured in IMDM medium containing fetal bovine serum, stem cell growth factor, thrombopoietin, and Fms-like tyrosine kinase 3 ligand during a 28-day ex vivo expansion process, while the expansion fold, specific growth rate, and the colony-forming efficiency were monitored. Then the Bmi1 and hTERT mRNA expressions in CD34+ cells were detected by fluorescence quantitative PCR, and the relations between the expressions and the cell proliferative capacity were analyzed. ResultsCD34+ cells expanded (20.1 ± 3.5) folds during the 28-day culture, while the proportion declined from 95.5% ± 2.6% before culture to 2.1% ± 0.4%. Both the specific growth rate and colony-forming efficiency of CD34+ cells declined significantly after 7 days, while the expression levels of Bmi1 and hTERT mRNA in CD34+ cells reached top at 7 days and declined gradually thereafter. ConclusionThe expressions of Bmi1 and hTERT genes may have a close relation to the proliferative capacity of CD34+ cells.
Objective To investigate the effect of hepatocyte-l ike cells induced by CD34+ cells in vitro on the repair of the injured hepatic tissues of mice in vivo. Methods Mononuclear cells were isolated from umbil ical blood by density gradient centrifugation and enriched CD34+ cells were obtained. The cells were (1 × 105 cells/mL) cultured in serumfreemedium containing stem cell factor (SCF), hepatocyte growth factor (HGF), EGF, oncostatin M (OSM), bFGF (the concentration were 50, 20, 20, 10, 10 ng/mL respectively) in vitro for 10 days. Forty-eight 6-week-old female ICR mice werechosen to prepare l iver injury model by injecting carbon tetrachloride and 2-acetylamionoflu-orene. The mice were randomly divided into two groups (n=24 per group): the experimental group, the cultured cells were injected into the mice through the tail vein; the control group, the equivalent serum-free medium was injected. Six mice from each group were killed at 7, 14, 21, and 28 days after operation to receive HE staining, PCR gel electrophoresis, immunohistochemistry staining, and hepatic function detection. Results HE staining: the morphology of injured hepatic tissues in the control group recovered to normal 28 days after operation, while in the experimental group, it recovered to normal 14 days after operation. PCR gel electrophoresis and immunohistochemistry staining: the cells expressing human serum albumin were detected in the hepatic tissue of the experimental group at each time point after operation; while in the control group, no such cells were detected within 28 days after operation. Hepatic function detection: the activity of alanine aminitransperase in the control group recovered to normal 14 days after operation; the mean activity of aspartate aminotransferase of two groups failed to recover within 28 days. Conclusion The hepatocyte-l ike cells induced by CD34+ cells in vitro can promote the morphological and functional recovery of the injured hepatic tissue in mice. Moreover, it can be transformed into human-derived hepatic cells in l iver-injured mice.
【Abstract】 Objective To observe the effect of cytokines and combinations in the inducement of human umbil icalcord blood-derived CD34+ cells into hepatocyte-l ike cells. Methods The mononuclear cells (MNCs) were derived by density gradient centrifugation and the CD34+ cells were sperated from MNCs. The human umbil ical cord blood-derived CD34+ cells were cultivated through 49 different combinations of cytokines including leukemia inhibitor factor (LIF), oncostatin M, bFGF, aFGF, hepatocyte growth factor, EGF and stem cell factor for 28 days, and the concentrations of the cytokines were 10, 10, 10, 10, 20, 20 and 50 ng/mL, respectively. The mRNAs of cytokeratin 19 (CK-19), CK-18, glutamine synthetase (GS), human albumin (ALB) and α-fetoprotein (AFP) were detected every seven days. The ALB secretion abil ity, detoxification abil ity and hepatin synthesis abil ity of the induced cells were detected by immunofluorescence assay, indocyanine green (ICG) and periodic acid-schiff assay, respectively. The fresh umbil ical cord blood-derived CD34+ cells were detected at the same time as a control. Results The mRNAs of CK-19, CK-18 and GS could be transcribed in all the induced cells, but the transcription of the mRNAs of ALB and AFP which was the special mark of mature hepatocyte and l iver stem cell, respectively, was not found. All the mRNAs could not be found in freshly isolated umbil ical cord blood-derived CD34+ cells. All the cells induced in vitro could not release ALB, and not help the detoxification of ICG which was the fundamental function of mature l iver cells. These results were the same in the control group. The hepatin synthesis abil ity of all the induced cells increased by comparison to the fresh ones. Conclusion Though some mRNAs of proteins which are transcribed in hepatocytes can be found in the induced cells, umbil ical cord blood-derived CD34+ cells could not be transdifferentiated into hepatocyte-l ike cells through cytokines in vitro.
Objective To investigate the expressions of vascular endothelial growth factor-C (VEGF-C) and its receptor Flt-4 in hepatocellular carcinoma (HCC), in order to analyze the relationships among their expressions, angiogenesis, lymph-genesis, and clinicopathologic features of HCC. Methods Sixty-two cases of HCC and 15 cases of normal hepatic tissue were studied with immunohistochemical method in order to inspect the expressions of VEGF-C and Flt-4, and to calculate the microvessel density (MVD) marked by CD34 and the lymphatic vessel density (LVD) marked by Flt-4. Besides their correlations, their relations with clinicopathologic features of HCC were further analyzed. Results The positive rates of VEGF-C and Flt-4 were obviously higher in HCC than those in normal hepatic tissue (Plt;0.05, Plt;0.01). The expression of VEGF-C in HCC was remarkably related with portal vein tumor emboli, histological differentiation of HCC, and postoperative recurrence and metastasis (Plt;0.05, Plt;0.01). The expression of Flt-4 in HCC was also related with histological differentiation and postoperative recurrence (Plt;0.05, Plt;0.01). MVD was related with tumor size, TNM clinical stage, histological differentiation, portal vein tumor emboli, and postoperative recurrence and metastasis (Plt;0.05, Plt;0.01). LVD was related with histological differentiation, postoperative recurrence and metastasis (Plt;0.05, Plt;0.01). Additionally, there was positive correlation between VEGF-C and Flt-4, MVD or LVD, Flt-4 and MVD or LVD, MVD and LVD, respectively (Plt;0.01). Conclusions VEGF-C and Flt-4 are highly expressed in HCC, and are related with postoperative recurrence, are positive correlated with MVD and LVD. It suggests that VEGF-C/Flt-4 might has an effect on progression and prognosis of HCC through promoting angiogenesis and lymph-genesis.
ObjectiveTo study the effects of the expressions of endostatin, basic fibroblast growth factor (bFGF) and CD34 on oncogenesis and progression of gallbladder cancer, and to explore some valuable criterias for its biotherapy. Methods The expressions of endostatin, bFGF and CD34 were studied by means of immunohistochemistry (SP) in 61 cases of gallbladder cancer and 10 cases of normal cholecystic tissue, and microvessel density (MVD) was calculated by the expression of CD34. Their relationships with clinical pathological features were also investigated. Results The expression rates of endostatin in normal cholecystic tissue and in gallbladder cancer tissue were 40.00% (4/10) and 77.05% (47/61) respectively, which had statistical difference (P＜0.05). The expression of endostatin in 61 cases of caner was relational to clinical stage and metastasis of lymph nodes (P＜0.05), while no significant correlation was detected with sex and age of patient, location of tumor, size of tumor and histologic grade (P＞0.05). The expression rates of bFGF in normal cholecystic tissue and in gallbladder cancer tissue were 20.00%（2/10） and 67.21% （41/61） respectively, which had statistical difference (P＜0.05). The expression of bFGF in 61 cases of caner was relational to clinical stage and metastasis of lymph nodes (P＜0.05), while no significant correlation was detected with sex and age of patient, location of tumor, size of tumor and histologic grade (P＞0.05). MVD in gallbladder cancer tissue and in normal cholecystic tissue was （76.66±20.15） piece/HP and (29.53±5.03) piece/HP respectively, showing significant difference (P＜0.01). In 61 cases of cancer, MVD in clinical stage Ⅲ～Ⅴ 〔(80.53±17.98) piece/HP〕 was much higher than that in stage Ⅰ+Ⅱ 〔(46.79±5.38) piece/HP〕, P＜0.01; MVD was higher in those with lymph nodes metastasis 〔(94.60±7.28) piece/HP〕 than those without metastasis 〔(58.12±9.24) piece/HP〕, P＜0.01; and MVD was (60.59±14.71) piece/HP in histologic grade G1， (83.08±15.30) piece/HP in G2， and (96.53±6.92) piece/HP in G3, the difference was significant among them （P＜0.01）. There was no significant correlation between MVD and sex and age of patient, location of tumor and size of tumor (P＞0.05). There were statistically significant correlations between expressions of endostatin and MVD （P＜0.01）, expressions of bFGF and MVD （P＜0.01）. Conclusions The result suggests that endostatin, bFGF and CD34 play roles in oncogenesis and progression of gallbladder cancer. Detection of these proteins has positive effects on diagnosis, malignant degree determination and treatment of gallbladder cancer.
Objective To observe the expressions of P53 and CD34 in rectal cancer and distal mucosa and to explore the safe distal margin of radical surgery for rectal cancer at molecular pathologic level. Methods Forty-five cases of rectal cancer were marked before operation， and then the cases were detected by PET/CT. P53 and CD34 expressions in rectal tissues were detected by immunohistochemistry technique. Results P53 expression and microvessel density (MVD) in rectal cancer were significantly higher than those in distal mucosa， which in distal mucosa were decreased along the anal direction. P53 and CD34 were still found in the normal rectal tissue. P53 expression and MVD were not significantly different between in more than 1.5 cm distal rectal mucosa and in normal rectal tissue. Besides MVD was related to size of tumor in rectal cancer and distal 0.5 cm rectal mucosa tissue, P53 and CD34 in rectal cancer and distal mucosa rectal tissue were not associated with tumor diameter, stage and differentiation of rectal cancer. Conclusion From the molecular pathologic view, the resection of 2.0 cm rectal distal tissue should be safe for excision of rectal cancer.
Objective To investigate the changes of indocyanine green retention rate at 15 minutes (ICGR15) of autologous peripheral blood CD34+ hematopoietic stem cells transplantation in end-stage liver disease (end-stage liver, disease, ESLD) patients with different Child-Pugh grades during before and after transplantation of 3, 6, 12, 36, and 60 months. Methods The CD34+ hematopoietic stem cells transplantation were performed in 60 cases of advanced liver cirrhosis with different Child-Pugh grades who were ineffectively treated with strictly conservative treatment and complied with the criterion of liver transplantation. The ICGR15 were performed before transplantation and in 3, 6, 12, 36 and 60 months after transplantation. And the results of each time point in each Child-Pugh classification group were compared, and the rate of change of ICGR15 value were compared between each Child-Pugh classification group. Results The ICGR15 values of the Child-Pugh grading groups all decreased with time. In Child A group, there were respectively significant differences between the 6 months, 12 months, 36 months, and 60 months groups after transplantation and preoperative and 3 months groups after transplantation (P<0.05), but there was no significant difference between preoperative and 3 months group after transplantation (P>0.05), and there was significant difference between the 12 months and the 60 months group after transplantation (P<0.05). As same as Child A group, there were also significant differences between that time groups in the Child B group (P<0.05), but there were also significant differences between the 3 months group after transplantation and preoperative (P<0.05), and there were respectively significant differences between the 6 months and 12 months, 36 months, and 60 months group after transplantation in the Child B group (P<0.05). Also in the Child C group, there were significant differences between that time groups (P<0.05), but there was no significant difference between preoperative and 3 months group after transplantation (P>0.05), and there were respectively significant differences between the 6 months and 12 months, 36 months, and 60 months group after transplantation (P<0.05). There was no significant difference in the rate of ICGR15 between Child-Pugh classification groups. Conclusion Autologous peripheral blood CD34+ hematopoietic stem cells transplantation can effectively improve the liver function reserve capacity of ESLD patients and improve the safety of operation for a long time.
Objective To explore the expression of CD34 and polyclone carcinoembryonic antigen (pCEA) of positive and negative alpha fetoprotein (AFP) detected by puncture biopsy in human hepatocellular carcinoma (HCC) and the significance of pathological diagnosis. Methods Fifty-four HCC tissue specimens from 2013 to 2015 were collected from tumor biopsy samples which confirmed by pathology in the Central Hospital of Enshi Tujia and Miao Autonomous Prefecture. The tissue samples were divided into positive AFP group (n=33) and negative AFP group (n=21) according to the detection results of serology and immunohistochemistry analysis of AFP. Expressions of CD34 and pCEA in the fifty-four HCC specimens were detected by immunohistochemistry. Results The positvie expression rate of pCEA in the positive AFP group was 69.7%, which was significantly higher than that in the negative AFP group (38.1%) (P<0. 05). However, the difference in positive expression rate of CD34 between the positive and negative AFP groups (90.91% and 85.71%, respectively) was not significant (P>0.05). Conclusion The associated detection of AFP, pCEA and CD34 in HCC tissues might contribute to the pathological and differential diagnosis of human hepatocellular carcinoma in puncture biopsies.
Immobilization of CD34 antibody on ferroferric oxide magnetic nanoparticles was achieved by the traditional carboxyl-amine conjugation reaction. Fourier transform infrared spectroscopy (FT-IR), nanoparticle size analysis (dynamic light scattering and transmission electron microscope), and other testing methods were used to detect the surface modified magnetic nanoparticles. The endothelial progenitor cells (EPCs) were cultured with the surface modified magnetic nanoparticles to evaluate cell compatibility and the combination effect of nanoparticles on EPCs in a short period of time. Directional guide of the surface modified magnetic nanoparticles to EPCs was evaluated under applied magnetic field and simulated dynamic flow condition. The results showed that the magnetic nanoparticles were successfully modified with CD34 antibody, which had good cell compatibility within a certain range of the nanoparticle concentrations. The surface modified nanoparticles can combine with EPCs effectively in a short time, and those nanoparticles combined EPCs can be directional guided under the magnetic field in the dynamic flow environment.
目的 检测血管内皮生长因子（VEGF）、白细胞分化抗原34（CD34）及CXC趋化因子受体4（CXCR4）在转移性鼻咽癌患者鼻咽部肿瘤组织中的表达，探讨它们与鼻咽癌各种临床病理因素的关系以及它们之间的相互联系。 方法 采用免疫组织化学链霉素抗生物素蛋白-过氧化物酶连结法检测2003年3月－2009年5月35例转移性鼻咽癌患者VEGF、CD34及CXCR4在鼻咽部肿瘤组织中的表达情况，结合患者临床病理特征进行分析。 结果 转移性鼻咽癌患者鼻咽部肿瘤组织中的VEGF及CXCR4阳性表达率分别为62.9%（22∕35）和42.9%（15∕35），CD34计数为11～92，平均43.2 ± 20.5。无肺转移较有肺转移的患者VEGF的阳性表达率高（78.9%、43.8%，P=0.043），多器官转移较单器官转移的患者CXCR4的表达强度高（62.5%、26.3%，P=0.044）。 结论 VEGF表达阳性的患者易发生肺转移；CXCR4强表达的患者易发生多器官转移。