Study On The Biological Characteristics Of The Intestinal Epithelial Cells Of The Ginseng

Objective:Study the general and special biological characteristics of intestinal epithelial cells(IEC)ofPheretima aspergillum(E.Perrier),establishthe test method and reference standard ofIEC,s biology characteristics;obtain the valuable experimental data about morphology,structure,metabolism and proliferation of IECin order toproviede asolid foundation forfurther study of DNA replication,transcription,protein synthesis,energy metabolism and cell proliferation.It also provides the necessary technical support for related research with other cells as material.Method:1.Morphology of second and third passage IECs in the logarithmic growth phase were observed by light microscope and cell images were captured using the software of View finder.To observe the ultrastructure of IEC,such as villi,organelles,nucleus was observed under an transmission electron microscope and its organelles wereaccurately classifiedwithreported literatureabout earthworm cell ultrastructures.2.Refering to the experimental method of mammalian chromosome karyotype analysis,chromosome karyotype analysis of ICE was carried out..The cells with clear shape whose chromosomes were not overlapped dispersed well under an inverted microscope were selected.By using Adobe karyotype model diagram were made using Excel after data of chromosome morphology were conducted in a standard mode by Photoshop 8.0 image software standard.The chromosomeswere named and classified according to the position of the centromere which Levan had put forwarded(1964).3.Respectively to broken preparing condition and dyeing liquid components of IEC were investigated based on the other similar animal cell cycle detection method.The advantages and disadvantages of four kinds of dying scheme were valuated with membrane breakage degree,cell cycle distribution histogram graphics,coefficient of variation(CV)and cellular Debris content Debris(%),the total number of cells(Cell No.)to determine and establish the optimum experiment conditions and scheme of detecting IEC Cell cycle.4.Stressed The second and third passage IECs in the logarithmic growth phase were induced by different concentration of Cd2+ treatment and incubatedfor0,12,24 and 48 hunder 37℃with 5%CO2.The IEC tolerance ability of heavy metals Cd2+ was evaluated usingthe morphological features,survival rate and vitality of ICE of Pheretima aspergillum.The experimental images and datawere analyzed using Microsoft Excel and SPSS17.0 software.Results:The study found that the diameterof IEC is about 8 ± 2 μ m,cellular morphology turn into round or oval fromcylindrical,villi appears the phenomenon of functional decline due to changes of environment.And,mitochondria,protein,endoplasmic reticulum and rough endoplasmic reticulum present specific distribution in space.2.Pheretima aspergillum IEC karyotype analysis show that Pheretima aspergillum is diploid organismdue to that it has 22 chromosomeswhich is 11 set of chromosomescontaining,as.Chromosome karyotype is expressed as n(♂)=x=6m+3sm+2st.IEC’sform appearsmorphological change when culturedin vitro,.but its morphology and number of chromosomes don’t presented obvious variation,which can be thought thatIEC genomewere relatively stableon epigenetic.3.Taken together with breaking condition of membranes,and the distribution of Cell cycle histogram graphics,CV value,content of cellular Debris and the total number of cells(Cell No.)to evaluate the optimum testing conditionsof IEC cycle,obtain a set of best dyeing solution:0.1%TritionX-100+10 μ g/mL PI+50μ g/mLRNase.The cell cycle in the histogram shows cellsin the Gl phase account for over 93%in the total cell number,which can objectively confirmIEC cultured in vitrohas long material preparation.In addition,the testing results of DNA content in the cell cycle confirmPheretima aspergillum hais diploid organismThe results are consistent with the chromosome karyotype.4.IECtolerancecharacteristicsof Cd2+was mainly as follows:①The highest concentration of Cd2+ IEC can tolerate was 8μmol/mL without incubation.②Treating with Cd2+ for 24h,the maximum tolerated concentration of IEC was6.25 x 10-2 μ mol/mL.③ Treating with Cd2+ for 48h,the maximum tolerated concentration of IEC was 3.125 × 10-2,mol/mL.The three concentrations were 2997.33、23.33、11.66 times of cadmium limit indicators ofDiLong according to "Chinese Pharmacopoeia"(2010 edition).④ IEC’ s tolerance of Cd2+ was negatively correlated with the dose in a certain time.Conclusion:The study obtain a set of morphological information related to the IEC and the general biological characteristicsfrom the most basic observation of IEC cells morphoalogical features toidentification ofe internal structure inadvanced level,.At the same time,,establishconditions and methods which is suitable for IEC chromosome karyotype analysis and thecell cycletest by selection optimization,master the detailed activity regularity of growth and reproductionof IEC cellin this paper,.In addition,collected important evidencethat the IECshave high tolerance to heavy metals Cd2+ and clearly understand the maximum tolerated concentrations of heavy metals Cd2+ and the law of stress reaction andtolerance ofIEC to provide valuable experimental basis forclarifingits enrichment mechanism of heavy metals at the cellular and molecular level.