Construction of the tissue engineering seed cells(HaCaT-EGF) and analysis of its biological characteristics

Objective:To construct the tissue engineering seed cell(HaCaT cell line) with stable expression of the human epidermal growth factor(EGF),and analyze the changes of its biological characteristics.Methods:PCDNA3.1-EGF eukaryotic expression vector was transferred into HaCaT cell,and G418 was utilized to select the HaCaT-EGF cell line.Using an inverted microscope,PCR,ELISA method to detect the changes of the cell morphology,the expression of the ECF gene and protein,and the mRNA expression levels of apoptosis related molecule Caspase-3,the cell cycle related protein cyclin D1.Results:The mRNA expression levels of the obtained HaCaT-ECF cell were more than 100 times higher than the level of ordinary HaCaT cell.The colony of the HaCaT-EGF cells was more focused and tight compared to the empty vector transfected HaCaT cells and normal HaCaT cells.The expression levels of apoptotic factor Caspase-3 and cyclin Dt in HaCaT-EGF cell were significantly higher than those in the empty vector HaCaT- pcDNA3.1 cell,and the differences were statistically significant(P<0.01),but there was no significant difference compared to the normal HaCaT cells(P>0.05).Conclusions: HaCaT-EGF cell can continuously secrete ECF,and the biological characteristic is stable.It can be used for tissue engineering experiment and is an ideal seed cell for constructing tissue engineered skin.

Two putative fatty acid synthetic genes of BgFas3 and BgElo1 are responsible for respiratory waterproofing in Blattella germanica

Water retention is critical for physiological homeostasis and survival in terrestrial insects. While deposition of hydrocarbons on insect cuticles as a key measure for water conservation has been extensively investigated, we know little about other mechanisms for preventing water loss in insects. Here, we report two fatty acid synthetic genes that are independent of hydrocarbon production but crucial for water retention in the German cockroach Blattella germanica (L.). First, an integument enriched fatty acid elongase gene (BgElo1) was identified as a critical gene for desiccation resistance in B. germanica;however, knockdown of BgElo1 surprisingly failed to cause a decline in cuticular lipids. In addition, RNA interference (RNAi)-knockdown of an upstream fatty acid synthase gene (BgFas3) showed a similar phenotype, and transmission electron microscopy analysis revealed that BgFas3- or BgElo1-RNAi did not affect cuticle architecture. Bodyweight loss test showed that repression of BgFas3 and BgElo1 significantly increased the weight loss rate, but the difference disappeared when the respiration was closed by freeze killing the cockroaches. A water immersion test was performed, and we found that BgFas3- and BgElo1-RNAi made it difficult for cockroaches to recover from drowning, which was supported by the upregulation of hypoxia-related genes after a 10-h recovery from drowning. Moreover, a dyeing assay with water-soluble Eosin Y showed that this was caused by the entry of water into the respiratory system. Our research suggests that BgFas3 and BgElo1 are required for both inward and outward waterproofing of the respiratory system. This study benefits the understanding of water retention mechanisms in insects.

Downregulation of NADPH-cytochrome P450 reductase via RNA interference increases the susceptibility of Acyrthosiphon pisum to desiccation and insecticides

Nicotinamide adenine dinucleotide phosphate(NADPH)-cytochrome P450 reductase(CPR)is involved in the metabolism of endogenous and exogenous substances,and detoxification of insecticides.RNA interference(RNAi)of CPR in certain insects causes developmental defects and enhanced susceptibility to insecticides.However,the CPR of Acyrthosiphon pisum has not been characterized,and its function is still not understood.In this study,we investigated the biochemical functions of A.pisum CPR(ApCPR).ApCPR was found to be transcribed in all developmental stages and was abundant in the embryo stage,and in the gut,head,and abdominal cuticle.After optimizing the dose and silencing duration of RNAi for downregulating ApCPR,we found that ApCPR suppression resulted in a significant decrease in the production of cuticular and internal hydrocarbon contents,and of cuticular waxy coatings.Deficiency in cuticular hydrocarbons(CHCs)decreased the survival rate of A.pisum under desiccation stress and increased its susceptibility to contact insecticides.Moreover,desiccation stress induced a significant increase in ApCPR mRNA levels.We further confirmed that ApCPR participates in CHC production.These results indicate that ApCPR modulates CHC production,desiccation tolerance,and insecticide susceptibility in A.pisum,and presents a novel target for pest control.