Mitofusion 2 Overexpression Decreased Proliferation of Human Embryonic Lung Fibroblasts in Acute Respiratory Distress Syndrome through Inhibiting RAS-RAF-1-ERK1/2 Pathway

Acute respiratory distress syndrome(ARDS)is one of the most fatal diseases worldwide.Pulmonary fibrosis occurs early in ARDS,and its severity plays a crucial role in ARDS mortality rate.Some studies suggested that fibroproliferation is an essential mechanism in ARDS.Mitofusion2(Mfn2)overexpression plays a role in inhibiting cell proliferation.However,the role and potential mechanism of Mfn2 on the proliferation of fibroblasts is still unknown.In this study,we aimed at exploring the effect of Mfn2 on the human embryonic lung fibroblasts(HELF)and discussed its related mechanism.The HELF were treated with the Mfn2 overexpressing lentivirus(adv-Mfn2).The cell cycle was detected by flow cytometry.MTT,PCR and Western blotting were used to investigate the effect of Mfn2 on the proliferation of the HELF,collagen expression,the RAS-RAF-1-ERK1/2 pathway and the expression of cycle-related proteins(p21,p27,Rb,Raf-1,p-Raf-1,Erk1/2 and p-Erk1/2).The co-immunoprecipitation assay was used to explore the interaction between Mfn2 and Ras.The results showed that the overexpression of Mfn2 inhibited the proliferation of the HELF and induced the cell cycle arrest at the G0/G1 phase.Meanwhile,Mfn2 also inhibited the expression of collagen I,p-Erk and p-Raf-1.In addition,an interaction between Mfn2 and Ras existed in the HELF.This study suggests that the overexpression of Mfn2 can decrease the proliferation of HELF in ARDS,which was associated with the inhibition of the RAS-RAF-1-ERK1/2 pathway.The results may offer a potential therapeutic intervention for patients with ARDS.

QUANTIFICATION OF P4HA2 mRNA OF FIBROBLASTS WITH SYBR GREEN BASED RT-PCR FOR CORRECTING CMV INACTIVATION EFFICIENCY IN DONOR BLOOD

Objective To quantify proline 4-hydroxylase, alpha polypeptide ii （ P4HA2 ） mRNA of human embryo lung fibroblast （HELF） with SYBR green based reversed transcript PCR （RT-PCR） for correcting cytomegalovirus （CMV） inactivation or clearance efficiency in donor blood. Methods A pair of specific primers of exon 12a of P4HA2 was designed, and the related PCR-reaction system and condition were optimized. Then the recombinant plasmid containing the target fragment was constructed for making standard curve with SYBR green based real-time RT-PCR. Finally, the sensitivity, reproducibility, and specificity of this method were fully estimated. Results The sensitivity of the method was 1.5E ＋ 04 copies/mL of P4HA2 mRNA, corresponding to 10^3 fibroblasts. In addition, existence of 8. 67E ＋ 06 leukocytes could not interfere with the accurate quantification of HELF in the large dynamic range. The intra-assay variability and inter-assay variability both varied in different concentrations, being higher in low concentrations and lower in high concentrations. But all of them were below 13. 76% in variation, which showed acceptable stability of this method. Conclusion SYBR green and specific primer based real-time RT-PCR show up a good quality for quantifying HELF P4HA2 mRNA with good specificity, stability, and high sensitivity. Approximate 10 copies of P4HA2 mRNA per cell in average can be detected by the method. Therefore, this method can be used to deduct fibroblast-associated CMV for correcting CMV inactivation efficiency in leukocytes.