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Objective:To investigate the correlation between the number of circulating DLBCL cell and the marrow tumor cell burden and the prognostic indicators in patients with DLBCL, and to evaluate the feasibility of circulating DLBCL cell reflecting the marrow tumor burden and disease progression. Optimization of FCM for screening circulating DLBCL cell was done to monitor MRD and recurrence.Methods:We conducted a retrospective study in 75 diagnosed DLBCL patients in the First Affiliated Hospital of Zhengzhou University from June 2020 to February 2021, including 43 males and 32 females aged 61 (37-85) years. According to the diagnosis and treatment criteria, the patients were divided into initial and recurrence group ( n = 53), partial response(PR)group ( n=14) and complete response(CR)group ( n=8). According to the positive criteria of circulating DLBCL cells, 48 cases were divided into circulating DLBCL positive group and 27 cases were negative group. 30 anemia patients with non-B-cell tumor-related diseases were selected as the control group, including 16 males and 14 females, aged 52 (30-79) years. 70 healthy subjects, including 36 males and 34 females, aged 39 (25-57), were selected for methodology optimization. FCM was used to detect the ratio of marrow and circulating DLBCL cells in each group, and analyze the connection between circulating DLBCL cells and clinical indicators. Statistical analysis was performed using t test, χ 2 test, Kruskal-Wallis H test, Spearman rank correlation, and Logistic regression. Results:(1) Bone marrow and circulating DLBCL cells were not detected in CR group and control group; The positive rate of circulating DLBCL cells in the initial/recurrent group and PR groups was 75.47% and 57.14%, respectively. The proportion of bone marrow and circulating DLBCL cells was positively correlated in the two groups ( P value was <0.001 and 0.020, respectively). (2) The proportion of bone marrow and circulating DLBCL cells in the initial and recurrent groups, PR group, CR group and control group decreased successively ( P<0.05). The proportion of DLBCL cells was 27.72% (initial and recurrent bone marrow group), 26.92% (initial and recurrent circulating group), 3.23% (bone marrow PR group) and 1.67% (circulating PR group), respectively. (3) Compared with the negative group, the circulating DLBCL cell positive group had increased LDH, β 2-MG, and CMYC expression(≥80%), with decreased LYM, HGB<100 g/L, B symptoms, PD-L1 expression, and age ≥60 years, showing higher ECOG, aaIPI/IPI scores and Ann staging ( P<0.05). Age ≥60, B symptoms, and PD-L1 expression were independent risk factors for circulating DLBCL cells ( P<0.05). Conclusions:The detectable rate of circulating DLBCL cell could be improved by optimizing the preoperative treatment conditions of FCM. Circulating DLBCL cells can reflect the tumor burden and disease progression. Detecting circulating DLBCL cells may improve patients′ compliance.
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Objective To setup a measurement of human bone marrow micromegakaryocyte which based on CD41a and PI double‐labeled flow cytometric analysis ,and study the significance in the diagnosis of MDS .Methods In 42 cases of MDS patients , their bone marrow megakaryocytes were obtained by Percoll density gradient separation medium .The megakaryocyte glycoproteinⅡb/Ⅲa(CD41a)were marked with fluorescein isothiocyanate through its corresponding monoclonal antibody ,and their DNA were marked with PI .Then the megakaryocyte ploidy was analyzed by flow cytometry(FCM ) .Results The method for micromegakaryo‐cyte identification and analysis was established .In 42 patients with MDS ,the detection rate of micromegakaryocyte was 90 .5 per‐cent by FCM analysis ,but only 54 .8 percent by Wright‐Giemsa staining test and 64 .3 percent by immunohistochemistry ,the differ‐ence among them was statistically significant(χ2 = 13 .640 ,P= 0 .001) .The 42 patients with MDS were divided into two groups (low‐risk group and high‐risk group) .The detection rates of micromegakaryocyte were 81 .8 percent in low‐risk group and 100 per‐cent in high‐risk group separately by FCM analysis ,the difference was statistically significant(χ2 =4 .019 ,P=0 .045) .Conclusion The detection rate of micromegakaryocyte by FCM with CD41a and PI double marker is higher than that by cytochemical staining . The detection rate of micromegakaryocyte in the high‐risk group is higher than that of the low‐risk group ,which shows that the de‐tection of micromegakaryocyte is of great significance for MDS prognosis assessment .
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Objective To investigate the relationship between aquaporin-1, -2, -3, -4 mRNA (AQP1-4) and renal parenchyma thickness in congenital hydronephrotic kidney in children. Methods The expressions of aquaporin 1, -2, -3, and -4 mRNA in hydronephrotic kidney of 37 children (aged 60.3±48.8 months) were evaluated with congenital hydronephrosis and control kidney of 6 children (aged 62.7±17.1 months) by using semi-quantitative reverse transcriptase polymerase chain reaction technique. Hydronephrotic kidney parenchyma thickness was measured by B-Ultrasound preoperative-ly and verified at operation. The relations of aquaporin 1, -2, -3, and -4 mRNA to the hydronephrotic kidney parenchyma thickness were analyzed by correlation analysis. Results The aquaporin 1 ,-2,-3, and -4/beta-actin ratio in the hydronephrotic kidney and normal kidney were 0.39±0.22 vs 0.90± 0.10, 0.42±0.20 vs 0.92±0.09, 0.525±0.22 vs 0.98±0.12, 0.30±0.18 vs 0.74±0.21 respec-tively, and the differences were significant (P<0.01). Hydronephrotic kidney parenchyma thickness measured by D-Ultrasound was 5.01±2.38 mm, which was identical with those measured at opera-tion. Significant correlation was found between the levels of aquaporin 1,-2,-3, and -4 mRNA and hydronephrotic kidney parenchyma thickness (r=0.773, 0.772, 0.557, 0.625, respectively; P< 0.01). Conclusions Significant correlation exists between decreased expressions of aquaporin 1 ,-2, -3, and -4 mRNA and atrophic change of renal parenchyma. This result may provide evidence to ex-plain the mechanism why the thinner renal parenchyma thickness, the weaker renal concentration and dilution function.
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Objective To study the role of the specific short hairpin RNA(NS-shRNA) of nucleostemin in inhibiting the expression of nucleostemin(NS) gene and evaluate the effect of NS-shRNA on differentiation antigen and biological properties of HL-60 cells,so as to explore the relationship between the biological functions of NS and acute leukemia,the potential perspective of NS gene therapy with RNA interference(RNAi).Methods HL60 cells were directly transfected NS-shRNA by using special transfection reagent.After 48 h,the inhibition rate of NS-shRNA to NS gene was detected by RTPCR,the proliferation ability of HL-60 cells was detected by MTT,the differentiation antigens were assayed by flow cytometry(FCM),the morphologic peculiarities such as cell shape,growth condition were observed,and the volume and granularity of HL-60 cells were analyzed by blood cell analyzer.Results The expression of NS gene were significantly inhibited by both two NS-shRNA,the inhibition rate were 37.82% and 71.88%,respectively.The significant inhibition effect of NS-shRNA to the proliferation of HL-60 cells existed in a time-dependent and concentration-dependent manner,and the best time was 48 h,the best concentration was 10 nmol/L.The change of differentiation antigen included increase of CD11b,CD33,CD14,CD64,HLA-DR and decrease of CD38,indicating continuous maturation of HL-60 cells to granulocytes and redifferentiation trend to monocytes.The aggregation of cells declined and the cell fragments increased.Myeloperoxidase(MPO) and ?-naphthol acetate esterase(?-NAE) activity increased after NS-shRNA-2 transfection.Furthermore,some HL-60 cells changed from round to fusiform shape even to pseudopod.The cells of small volume and karyorrhexis increased. Conclusion Through blocking the expression of NS gene,NS-shRNA can inhibit the cell proliferation and induce the differentiation of HL-60 cells,weaken malignant degree of HL-60 cells and trigger cell apoptosis.NS-shRNA is of clinical potential in gene therapy for acute leukemia.
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Aim In order to further explore the feasibility of gene therapy with RNA interference(RNAi) for acute leukemia,we synthesized short hairpin RNA(shRNA)for nucleostemin(NS)in vitro.Methods The designed DNA template consists of the sequence complementary to target mRNA,which was screened out consensus motif of three variants of NS gene,using 5′-UCUCUUGAA-3′ as loop sequence,The sequence of 5′-GGATCCTAATACGACTCACTATA-3′ acts as T7 promoter.Two shRNA were produced by T7 RNA polymerase and named as shRNA-NS-1 and shRNA-NS-2,respectively.The purified shRNA was quantified by gel electrophoresis.The interference effect of shRNA-NS transfected into HL-60 cell was examined by resultant cell morphology and inhibition rate of NS-mRNA expression.Results The concentrations of two shRNA-NS without degradation and diffusion were 5.24 ?mol?L~(-1)and 3.35 ?mol?L~(-1),respectively.There were significant decline in density and aggregation of cells and significant differences in size between cells after interfering HL-60 cell for 48 h.Furthermore,some of HL-60 cells treated by shRNA-NS-2 were changed from round to fusiform shape even with pseudopod.Compared with control group,the inhibition rates of shRNA-NS-1 and shRNA-NS-2 to NS-mRNA expression were 37.82% and 71.88%,respectively.Conclusion The two shRNA for NS gene were successfully constructed,which suppress NS gene expression significantly.shRNA-NS-2 also can chang HL-60 cell′s morphology and might be a useful tool to explore NS gene function and possible therapy for acute leukemia.