The Relationship Between Overloading Of [Ca～(2+)]i In SO Cells Of Rabbit With Hypercholesterolemia And The Gene Expression Of Kca Channel And The Molecular Cloning Of Relevant Gene

Background and objective: It had been proposed that the sphincter of oddi dyskinesia or pathological changes were the potential causes for stomachache, fever and jaundice in some patients when Oddi firstly found the Sphincter of Oddi (SO) in 1887.After that, many scholars raised these concepts ,such as papillitis , papillary stenosis , papillary stenosis , sphinctesr of oddi dyskinesia as well as phincter of oddi dysfunction(SOD) on the base of clinical observation, x-ray examinationand operation reviews. The modern concept of SOD is definitely defined as a kind of syndrome including repeatedly stomachache, fever, jaundice and a raised diastase level,bile stasis or pancreatitis caused by the poor bile or pancreatic juice drainage. But SOD haven't been accepted widely because of lacking effective diagnosis methods. With the development of ERCP technique (Endoscopic Retrograde Cholangio_Pancreatography), there is a new way to guarantee the correct diagnosis of SOD by sphincter of Oddi manometry (SOM). Now sphincter of Oddi manometry and ERCP are golden indexes of SOD.Although the mechanisms of SOD have been explored for manyyears,none of them could provide a persuasion analysis.Since 1990s,our program group have been investigating the mechanisms of SOD progressively . The progress of stuy are synchronous with that of scientists abroad have acquired now and take a lead in domestic. We have finished a series of researches and obtain several study achievements including 1 SOD clinical and lab experiment in human being and Canine ;2 the study of cholesterol liposomes influence on kinesis function of rabbit SO cells;3 the observation of SO ultrastructure and kinesis function in SOD rabbit models with hypercholesterolemia;4 the relationship among SOD , gallbladder bile stasis and the formation of cholelith in rabbit with hypercholesterolemia;5 the experiment requirements for SO cells laser scanning cofocal micrographs and recording of Ca²⁺ sparks and Ca²⁺ transmembrane movement in SO cells;6 the analysis of Ca²⁺ homeostasis characters and transmembrane movement in rabbit SO cells. All of these results have testified our study strategy of SOD is right.One of the most important research findings from our studies of SOD mechanism is the fact that Ca²⁺ metabolic disorder in SO cells .It is present as Ca²⁺overloading in SO cells, the lower velocity of Ca²⁺transmembrane movement, abnormalities in the hyperpolarization process of SO cell membrane . We can propose that Kca channel is the key site for study the mechanism of SOD as an important downstream site in regulating [Ca²⁺]i.The study results known revealed that it has a close relationship between the β subunit average amount/ per channel and Kca channel function ,in other word ,the changes in Kca channel function is the consequences of the stoichiometry of α and auxiliary β subunits. Untill now ,there have not any research results about hypercholesterolmia and thestoichiometry of α and auxiliary β subunits^the components of Kca channel.To explore the changes of the stoichiometry of α and auxiliary β subunits of Kca channel, we detected the expression level of α and β subunit on both mRNA transcription level and protein expression level by semi-quantity RT-PCR and semi-quantity Western Blot. We analysised the relationship between the imbalance feedback mechanism of Kca channel and the expression level of channel subunits, as well as any changes on DNA level inhypercholesterolemia condition. Main methods and Results 1. Preparation of polyclonal antiserum against β1 subunit of rabbit Kcachannel.Gene encoding the intracellular fragment of rabbit Kca channel's β1 subunit was amplified by RT-PCR. The GST- β1 fusion protein was expressed in E. coli. The fusion protein from PAGE gel was used to immunize BALB/c mice and prepared polyclonal antiserum. The specificity of antiserum was identified by ELISA and Western blot.a. A unique band about 300 bp was amplified by RT-PCR., which encoding the intracellular fragment of rabbit Kca channel's β1 subunit The amplified sequence consisted with the published one shown by sequence analysis.b. The SDS-PAGE analysis showed that the Mr of the fusion protein GST-β1 was about37 000. The purity of GST-β fusion protein was over 95%.c.The polyclonal antiserum against GST- β fusion protein could recognize both GST- β and β proteins in rabbit tissues. The highest titer of the antiserum was about 1:128 000, as shown by Western blot and ELISA,respectively.2 Preparation of guinea pig polyclonal antiserum against α subunit of rabbit Kca channel.Gene encoding the B cell epitope of rabbit Kca channel's α subunit was amplified by RT-PCR. The hisαa fusion protein was expressed in methylotrophic yeast Pichia pastoris and purified. The guinea pig polyclonal antiserum.against α subunit of rabbit Kca channel was acquired by immunize guinea pig with DNA vaccine.he specificity of antiserum was identified by ELISA and Western blota .Gene encoding the B cell epitope regions of α subunit of rabbit SO cell BK channel was amplified by RT-PCR, which is about 1 497bp. The amplified sequence consisted with the published one shown by sequence analysis.b .The SDS-PAGE analysis showed that the M_r of α subunit product was about 55 000. The concentration of the fusion protein was 55 mg. L^-1 and the purity was 96%.c .The guinea pig polyclonal antiserum against α subunit of rabbit Kca channel could recognize both his-α and a proteins in rabbit tissues. The highest titer of the antiserum was about 1:64 000, as shown by Western blot and ELISA, respectively.3 The study of mRNA transcription level and protein expression level of α and β subunit of rabbit Kca channel.TO detect the changes of mRNA transcription level and protein expression level of α and β subunit of rabbit Kca channel with semi-quantitative RT-PCR assay and semi-quantitative Western blot in 10 rabbit SOD models and normal controls.a.There is no statistic difference in the mRNA transcription level of P lsubunitof rabbit Kca channel in rabbit SO cells with or without SOD [SOD vs.control : (0.1886 ± 0.0559) vs (0.1851±0.0777), P =0.9118 >0.05]b .A significant raise in mRNA transcription level of a subunit of rabbit Kcachannel with SOD was observed [SOD vs. control : (0.4503 ± 0.1864) vs(0.2623 ± 0.1002), P =0.0154<0.05]c .Compared with the control group, β 1 /α were approximately 37%less abundant in SOD group in the mRNA transcription level. [SOD vs.control : (0.4456 ± 0.1471) vs (0.7139 ± 0.1806), P =0.0024<0.05]d We found no difference in the relative abundance of α and β 1 translationlevel[P_α=0.0899>0.05; P_β1 = 0. 6674 >0.05]e. In sharp contrast, the stoichiometry of auxiliary β lsubunits and α in theprotein expression level reduced about 35% in SOD group [SOD:0. 8357± 0.1583; control: 1.2962± 0.4273; P =0.0054<0.05]4 Sequence determination and analysis of β lsubunit of rabbit SO cellKca channel using rapid amplification of cDNA ends.In order to acquire the full-length genomic sequence of rabbit SO cell Kca channel and to analysis the way through which hypercholesterolemia regulated Kca channel transcripton and translation level as a environmental agent, the first time the full-length cDNA of β lsubunit gene of Kca channel was cloned from rabbit SO cell using RACE-PCR.a. A unique band about 1 169 bp was amplified by RACE -PCR., It contains an ORF about 573 bp, which encoding 191 amino acid sequence.The 5'UTR region of this gene has 92— 165bp more than that of rabbit other tissue P lsubunit gene of Kca channel.b. Sequence analysis shown that the gene fragment cloned has 85% and 86...