Hydroxyapatite Nanoparticles-mediated In Vitro & In Vivo Gene Transfection Of Rat Hepatocytes

Experiment I Transformation, extraction and identification of pEGFPC1Objective: To prepare the plasmid pEGFP-C1 for gene transfection.Methods: The competent E.coli JM109 were prepared chemically through CaCl₂ method and transformed with pEGFP-Cl, then the transformed bacteria stock were amplified by incubation in selective medium containing Kanamycin. With alkaline lysis method, plasmids were extracted on a small scale and the bacteria colonies with plasmids correctly corresponding to the digestion of restriction endonuclease EcoR I were incubated on a large scale. Plasmids were confirmed again through EcoR I digestion and measured for quantity and purity with spectrophotometer.Results: The experiment group with transformation of pEGFP-Cl formed colonies on agar plate containing Kanamycin, but not for the control group without transformation. The extracted plasmid could be digested by EcoR I to produce a fragment of 4.7kb identical with the length of pEGFP-Cl. The OD₂60/280 ratios of all the extracted plasmids ranged from 1.8 to 2.0 and the concentration ranged from 0.87 to 1.75μg/μl.Conclusions: 1. Competent bacteria can be prepared with highefficiency through CaCl2 method. E.coli JM109 transformed with pEGFP-Cl expresses the property of Kanamycin-resistance. 2. The extracted plasmids were confirmed to be pEGFP-Cl according to EcoR I digestion analysis. 3. Alkaline lysis method is a reliable, efficient procedure for plasmid extraction. Quantity and purity of pEGFP-Cl extracted with alkaline lysis method meet the requirement of gene transfection.Experiment II Preparation, surface modification and DNA binding of hydroxapatite nanoparticlesObjective: To prepare hydroxapatite nanoparticles modified with poly-L-lysine(PLL), and investigate the capability of hydroxapatite nanoparticles(HANP) in absorbing and protecting plasmid DNA.Methods: HANP were synthesized using the homogeneous co-precipitation method. The resulting HANP were modified with PLL in neutral environment, in neutral environment after being treated by Na2CO3, in alkalescent environment and in alkalescent environment after being treated by Na2CO3, respectively. The cationically modified HANP were tested for capacity of binding and protecting DNA by agarose gel electrophoresis and measuration of DNA concentration in supernatantafter centrifugation.Results: Under transmission electron microscope, the synthesized product presented needle-like and well dispersed particles with evenly distributed sizes of 15" 20nmx60 80nm. Treated by Na2CO3, HANP were effectively modified with PLL in alkalescent environment. PLL-HANP/DNA mixtures with w/w ratios upwards of 30 .' 1 effectively binded DNA and protected it against being digested by DNase I .Conclusions: 1. Through homogeneous co-precipitation method, HANP can be synthesized with needle-like figure and small, evenly distributed sizes. 2. Under alkalescent condition, the Na2CO3 treated HANP can be well surface-modified with PLL. 3. PLL- HANP are able to electrostatically bind and protect DNA.Experiment DI PLL- HANP-mediated in vitro gene transfection to the rat hepatocytesObjective: To investigate the capability of PLL- HANP as a carrier for in vitro gene transfection.Methods: Rat liver regeneration model was established by partial hepatectomy(70%), 2 days late, hepatocytes were isolated by two-step collagenase technique and purified by pecoll density gradientcentrifugation followed by determination of cell viabilities with trypan blue exclusion assay. The potential cytotoxicity of PLL-HANP on cultivated rat hepatocytes was measured by MTT assay. When cultivated to 60-70% cell confluence, hepatocytes were transfected with pEGFP mediated by PLL-HANP and liposome, respectively. Transient expression of EGFP was observed with fluorescent microscope 48 hours after transfection and transfection efficiencies of the two gene carriers were assessed.Results: The residual hepatic lobes enlarged 2 days after establishment of Rat liver regeneration model. Harvest of hepatocytes yielded 1.4 xlO8 cells for one rat with a cell viability of 95% after isolation and purification. MTT assay showed no evident hepatocytic toxicity for PLL-HANP when compared to liposome. Fluorescence could be detected by fluorescent microscope in PLL-HANP group and liposome group. The mean transient transfection rate of EGFP in PLL-HANP group was 26.8% , slightly higher than the rate of 24% in liposome group, but without significant difference to the latter(P>0.05).Conclusions: l.Rat liver regeneration model can be established by partial hepatectomy(70%). 2. hepatocytes can be harvested with high purity and viability through techniques of two-step collagenase perfusion and pecoll density gradient centrifugation. 3. PLL-HANP is safe for application to cultivated hepatocytes 4. PLL-HANP can mediateefficient gene transfection in vitro, is hopeful to become a promising carrier for gene transfection. 5. As a live cell reporter, EGFP is an ideal marker for detecting gene transfection.Experiment IV PLL- HANP-mediated in vivo gene transfection to the rat liverObjective: To investigate the feasibility of PLL- HANP as a gene carrier for in vivo gene transfectionMethods: Rat liver regeneration models were established by partial hepatectomy(70%), and 24 hours later PLL-HANP-pEGFP and liposome-pEGFP were injected into rat livers via portal vein with hepatic isolation. Hepatocytes were isolated by two-step collagenase technique and purified by pecoll density gradient centrifugation 4 days after transfection. Characterization of transient EGFP expression was observed with fluorescent microscope and transfection efficiencies of the two gene carriers were analyzedResults: Harvested hepatocytes ranged from 1.07xl08 to 1.62xlO8 cells per individual rat and the cell viabilities were determined by trypan blue exclusion assay to be around 95%. Green fluorescence exhibited by EGFP-transfected hepatocytes could be detected under fluorescentmicroscope in PLL-HANP group and liposome group. The mean transient expression rate of EGFP in PLL-HANP group was 20.2% slightly higher than the rate of 17.4% in liposome group, but without significant difference to the latter(P>0.05).Conclusions: 1. Based on establishment of the rat liver regeneration model, in vivo liver gene transfection through the procedure of portal venous injection with hepatic isolation can be achieved. 2. PLL-HANP can mediate gene transfection in vivo, may possibly become a promising carrier for in vivo gene transfection...