Effect Of Human Cytomegalovirus To The Expression Of HOXB6,HOXA5 Homeobox Gene On The Proliferation Of Granulocyte-macrophage Progenitor In Vitro

Abstract: objective: The objective is to observe the expression of HOX B6 gene , HOX A5 gene on the differentiation and proliferation of hematopoietic stem-progenitor cell to colony forming unit-granulocyte-macrophage .And the differentiation progress affected by HCMV and/or ATRA, aimed to investigate 1.Wether HCMV can lead to malignant blood desease is related to the regulation of homeobox genes. 2 . Wether the reason ATRA has the cure effect of malignant blood disease , especial acute nonlymphocytic leukemia (M3a) is related to the regulation of homeobox genes. 3.How the HOX B6 . HOX A5 regulate the hematopoietic lineage determination and maturation, especial myelopoiesis. Methods : 1. 10 cases cord blood samples were offered by obstetric of the affiliated hospital, all samples were collected from fetal placenta umbilical vein. 2.Groups: including 4 groups. (1) Normal CFU - GM culture was used as blank control (2)HCMV of 10^-4.5/0.1ml tissue culture infectious ( TCID₅₀ ) was diluted to 100TCID₅₀ 0.1ml, the diluted HCMV fluid was added into normal CFU- GM colonies culture system directly as HCMV infected group (3) ATRA (60nmol/ 1), was added into normal CFU- GM colonies culture system directly as HCMV infected group (4) HCMV and ATRA were added into culture system together, final concentration as above. 3. By the colony culture in vitro, the impact ofHCMVAD)69 ? ATRA on the CFU-GM colony formation were surveyed.;Observe the expression of H0XB6, H0XA5 gene on the differentiation progress of Hematopoietic Stem-Progenitor Cell (HSPC) to CFU-GM affected by HCMV and/or ATRA on the third, seventh , and twelfth day. 4.Mononuclear cells were isolated by Ficoll-Hypaque density centrifugation and cultured as the above system , gathered respectively at day 3^ 7> 12, washed in 1 X phosphate-buffered saline (PBS) prior to cryopreservation at -80°C in 90% fetal bovine serum (FBS) 10% dimethyl sulfoxide (DMSO). Gathered cells were rapidly thawed in a 37°C water bath, diluted in warm IMDM media containing 10% FBS, washed (x 2) with IMDM, and then pelleted for RNA isolation. 5. Total RNA was isolated by the guanidinium thiocyanate/acid phenol method using Trizol reagent in accordance with the manufacturer's standard method. RNA pellets were washed with 70% ethanol containing diethyl polycarbonate (DEPC), air dried, and redissolved in 0.01% DEPC by heating at 55°C. The integrity and purity of the RNA was assessed by agarose gel electrophoresis. Following the quality assessment of the RNA, aliquots were quantitated by absorbance at 260 and 280 nm. 6. Real-time quantitative PCR (RQ-PCR) : RQ-PCR was carried out using TaqMan probe-based chemistry. This chemistry provides for a high level of specificity through the design of complementary oligonucleotide primers and 5'-reporter/3' quencher fluorogenic probes. During the normal PCR process the fluorogenic probe is cleaved by the native 5'-exonuclease activity of TAQ polymerase that releases the reporter from the quencher, resulting in the generation of asequence-specific signal. Each additional cycle results in the release of reporter molecules from the respective probes. The fluorescence intensity is related to the initial number of RNA copies, which can be assessed by determining the threshold cycle (CT). All HOX-specific primers and probes were designed against GenBank-published sequences in association with Primer Express (Applied Biosystems). Endogenous controls were purchased as RNA-specific Pre Developed Assay Reagents (PDARs;Applied Biosystems) 7 .The amplification reactions (12.5 fXL) contained 50 ng cDNA equivalents (or control), 1 x Taqman universal PCR master mix. 8. Amplifications were performed following an initial 2-minute incubation at 55°C to allow uracil-N-glycosylase (UNG) to destroy any contaminating RNA, followed by treatment at 94°C for 10 minutes to inactivate the UNG enzyme and activate the DNA polymerase. This was followed by seted cycles . An FTC 1000 Sequence Detection System equipped with a 96-well thermal cycler was used for the amplifications. Data were collected and analyzed with Sequence Detector vl.6.3 software (Applied Biosystems). 9. The component of the colonies of myelomonocytic progenitor was determined by Giemsa -Wright staining. 10. Statistical methods:The results were showed by means plus or substracting standard deviation and increment or inhibition ratio Carry through homogene tity of variance ano one—way Anova After having statistics meaning.we compared means between groups by LSD and SNK .All these accomplishtics software spss 13.0. Results: l.Homeobox genes (HOX) do have a regulatory function in the differentiation process of hematopoiesis. During the differentiation and proliferation ofhematopoietic stem-progenitor cell to colony formingunit-granulocyte-macrophage in vitro, the expression of HOX A5 was almost negative while the expression of HOX B6 was significant positive . 2. Compared with the expression of H0XB6 on day 3 , the quantity of HOXB6 was obviously higher on day 7 and lower on day 12 respectively in each group. 3. The differentiation progress affected by HCMV and ATRA. Compared with the expression of H0XB6 on of normal group, the expression of H0XB6 of the group ATRA were up-regulated remarkablely (p<0.05), HCMV can down-regulate the expression of H0XB6. ATRA can against the down-regulated effect caused by HCMV as we can see from the test that when normal group interfered by ATRA and HCMV together, compair the HCMV group ,the already down-regulated expression of H0XB6 was increase again. Conclusion : Homeobox genes (HOX) do have a regulatory function in the differentiation process of hematopoiesis. The HOX A5 gene perhaps was not the key gene during the differentiation and proliferation of hematopoietic stem-progenitor cell to colony forming unit-granulocyte-macrophage in vitro. Homeobox gene B6 significantly play a important role in the process which can be concerned the HOX B6 regulate the hematopoietic lineage determination and maturation, especial myelopoiesis. 60nmol/ml ATRA can up-regulate the expression of HOXB6. At the same condition , HCMV (TCiD5045 /0.1ml) 100TCID5() 0.1ml can down-regulate the expression of HOXB6 and lead a suppression effect on the cell morphology , which confirmed the theory that the normal hematopoietic lineage determination and maturation rely on the stableand consistently expression of Homeobox genes. The fact that ATRA can against the down-regulated effect caused by HCMV hints the regulation mechanism of ATRA and HCMV to the Hox gene was related .