Construction Of Eukaryotic Expression Vector Of The Gene SLC34A2 And Experimental Study Of Human Lung Alveolar Epithelial Cell Transfected

This review summarizes the characteristics of the solute carrier familySLC34 that is represented by the type ll Na/P(i)-cotransporters NaPi-lla(SLC34A1), NaPi-llb (SLC34A2) and NaPi-llc (SLC34A3).The geneSLC34A2 encode Na+, phosphate contransporter(NaPi-IIb) and express in mangtissues of the human such as lung, intestines, kidney, thrroid, epiclidymis. All ofabove, the lung tissue is most. The phosghate balance is regulated by geneSLC34A2, which is regulated by kinds of hormones, low-phosphate diet, PH,and so on. The gene SLC34A2 is correlate to Pumonary AlveolarMicolithiasis(PAM), Testicular Microlithiasis, Primarily Intrahepatic Bile DuctMicrolithissis and cinosty organ microlithiasis. Pumonary AlveolarMicolithiasis is an not-know reason rare disorder in which microliths are fromand in the alvolar space. October in 2006 the researcher of Turkey identified aPAM locus by hemocygosty mapping to 4p15, then identified by a candidategene approach. The gene responsible for the disease as SLC34A2 which isinvolved in PAM. Then researcher from Japan also identified mutation in theSLC34A2 in patients of PAM. At present, the abroad researcher study the expression of the SLC34A2 inintestines and Coco cells mostly. There is not report about lung and alveolarepithlial cell.To study the function of the SLC34A2, especially in lung and alveplarapithelial cells, we clone the cDNA of the SLC34A2 and constructe theeukaryotic express which is transfected to alveolar epithelial cell, then thefuction of the gene is identified. At last, we find and treat a family of PAM anddetect whether the SLC34A2 mutation in this PAM family or not. Our researchsupply the rationale of the kind of disease and treat method. This researchestablish the important experimant foundation for studying the fucntion andmechanism of the gene SLC34A2.一,ConstructiConstruction and Identification of Eukaryotic Expression RecombinantVector of TypeⅡb Sodium Phosphate Cotransporter SLC34A2 GeneTo construction and Iidentification of eukaryotic Eexpressionrecombinant vector of typeⅡb Ssodium phosphate cotransporter SLC34A2gene, we find human SLC34A2 cDNA complete sequence from GenBank, thenprimers were designed. Reverse transcription-polymerase chain reaction (RTPCR)was used to amplify the SLC34A2 gene . The fragment was subclonedinto appropriate site of pcDNA3.1 (+) eukaryotic expression vector byrestriction enzyme digestion and linking reactions. The positive recombinantwas identified by restriction endonuclease digestion, PCR amplification andsequencing. Results: The SLC34A2 gene (2073bp) is successfully amplifiedfrom normal human lung tissue, and Restriction enzyme analysis andsequencing showed that the eukaryotic expression recombinant pcDNA3.1(+)-SLC34A2 was successfully constructed. 二,The study of fuction of the human lung alveolar epithelial cell A549 that istransfected by the gene SLC34A2The mRNA and NaPi-Ⅱb express in all kind of tissues, especially in lungand trachea, the NaPi-Ⅱb regulate the balance of calcium and phosphate inextracellular fliud. We have cloned the gene cDNA from the normal lung andconstruct eukeryotic expression recombinant vector.In this study, SLC34A2 is transfected to A549 cells via lipid body anddetect the expressionof SLC34A2 by RT-PCR, observe the change of calciumand phosphate in extrcellular fliud when SLC34A2 is overexpressed. Otherwise,we observe the expression of SLC34A2 and the change of calcium andphosphate cells supernatant when the A549cells are stimulated by different drug.Result: After pcDNA3.1 ( + )-SLC34A2 was transfected for 24 hours, theresult of RT-PCR show: the electrophoresis strip that we got is the same to thatdesigned. Then we analyssised the photodensity of the the SLC34A2 gene andintro-reference GAPDH by image analysis system, the relative expressioncontent the gene detected is the ratio of the gene detected and GAPDH. Theresult of half-quantitation is pcDNA3.1(+)-SLC34A2 was successfullytransfected and the amount of expression of group transfected is obviosly higherthan control and blank(P＜0.05). The change of calcium and phosphate in cellsupernatant is also that(P＜0.01), there was not significantly differencebetween control and blank group(P＞0.05). These result showed: The contentof Calcium and Phosphate decreased with increasing of the expression ofSLC34A2 in A549 cells, that was ,the gene SLC34A2 promoted the translationof Calcium and Phosphate from extracellular to incellulae.The result of SLC34A2 expression after the cells was interfered by glucocorticosteroid and estradiol: the high dose dexamethasone promote theexpression of SLC34A2, there was significantly difference compare withcontrol group(Q=3.2703 P<0.05), there was significantly difference in groupcomparison of different dose(P <0.01), the middle ang the low dosedexamethasone inhibited the expression of SLC34A2, there was significantlydifference compare with control group(Q=8.6384 P <0.01,Q=22.5481 P<0.01). The high and muddle dose estradiol inhibited the expression ofSLC34A2, there was significantly difference compare with control group andblank group(Q= 22.2928 P <0.01,Q=23.8244 P <0.01), there wassignificance difference in goup comparison of different dose(P <0.05), therewas not significantly difference between the low estradiol group and controlgroup(Q=1.5316 P >0.05). These result show: In A549 cells, high doseglucocorticoid can up-regulate the expression of SLC34A2, on the contray,estrogen may be inhibitor of SLC34A2.The content of Ca and P in cells supermatant after A549 cells wasinterfered by glucocorticoid: There was significantly difference between thehigh dose glucocorticoid and control group(P <0.05), there was notsignificantly difference between the other dose group and contol group(P>0.05); There was significantly difference between the low dose estradiolgroup and contol group(P <0.01), there was not significantly differencebetween the other dose group and contol group(P >0.05). These result show:high dose glucocorticoid can increase the expression of SLC34A2 and promotethe translation of Calcium and Phosphate from extracellular to incellulae. Thehigh dose glucocorticoid may be excitomotor of SLC34A2,which can directclinical work. 三,PulmonarPulmonary alveolar microlithiasis: four cases of a family report anddetection of the gene SLC34A2There a new report show recently that the mutation of SLC34A2 inducePAM. The scholar from Turkey and Japan detect the mutation of SLC34A2 inthe patient of PAM. We found an typical family of Pulmonary alveolarmicrolithiasis . In this study we analysis the charaterstic of clinical data of thefamily and detect whether there was mutation or not in the family.Result: There was 18 member in their four generations, grandparents ishealthy without specific disease history; parents is healthy ,then they marry asclose relative, there was no abnormalities in their health examination and CT;there four patients in third generation; these patients'child is healthy. Therethree of four patients was foud by health examination, the"3"case wasdiagnosed due to spontaneous pneumothorax and his symptom was typical:cough gradually, accelerated breathing and obviously after exertion. Physicalexamination: achropachy, cyanosis of oral lip, distention of jugular vein,Bp=150/90mmHg, HR=107vices/min, cardiac rhythm regular, not patho-souffle,breath sound of two lungs coarse, Velcro,bubble of two lower lungs, bloatedof both lower extremities. The CT of chest show: there trabs shadow in lobusinterior pulmonis and conferted microlithiasis shadow, bullae can be seen.Respiratory function: rule pulmonary ventilation function is mixed typeobstacle(midrange confined type), small airway function is serious blockedobstacle, maximal ventilatory volume decrease obviously. The hypersoundexamination of heart show: cor dextrum bigger, atrioventricular valve backflow. Electrocardiogram show: sinus tachycardia, right axis deviation, normalelectrocardiogram, pneum-P wave, clockwise rotation. Serum sodium 154mmoI/L,serum chlorine 110 mmoI/L,serum calcium 3.00 mmoI/L,serum phosphate 1.51 mmoI/L；belly color hypersound: calcif intrahepatic bile duct.blood gas analysis: pH 7.34,BE -8.6 mmol/ L,BB 38.1,PCO2 25.9 mm Hg,PaO 34.9 mm Hg,SO2% 71%. The patient show dyspnea gradually aggravated,was given absorb oxgen, diureses, glucocorticoid treatment after in hospital,the symptom of dyspnea was improve obviously, then CT and objective signwas not improve.The patients of this family have typical clinical character and the level ofserum ion was disorder, serum sodium and calcium were higher than healthgroup, that was different to thiose reported before. We need more researchs toprove whether the result of pathogenetic condition developed or pathogenesyof the PAM. Glucocorticoid treatment to The case"3'was effective, that isconcordant with the result of the experiment two, more study need to done toprove this result. In this study, we got nine extrons, seven extrons weresequenced successfully. We did not find mutation of SLC34A2, however, weestablished experiment foundation to study the gene again and accumulatedprecious clinical data.In a word, in this study the SLC34A2 gene (2073bp) is successfullyamplified, and the eukaryotic expression recombinant pcDNA3.1 (+) -SLC34A2 is successfully constructed first time. The positive recombinant wasidentified by restriction endonuclease digestion, PCR amplification andsequencing. The pcDNA3.1 (+)-SLC34A2 is transfected successfully to A549cells via lipid body. We proved that the SLC34A2 was overexpressed in cells byRT-PCR, the overexpression of the gene encouraged calcium and phosphate totransfer from extracellular fluid to intracellular fluid；this study showed that inthe human lung alveolar epithelial cell A549, high dose glucocorticoid promotethe expression of SLC34A2 and transferation from extracellular fluid to intracellular fluid of calcium and phosphate. The low dose glucocorticoidinhibited the expression of SLC34A2. The estrogen can not promoteexpression of SLC34A2, the high dose estrogen inhibited the expression ofSLC34A2. We found, treat and pursuit a familly of PAM, got firsthand clinicaldata, seven extrons were sequenced successfully firstly internal. establishedexperiment foundation to study the gene and pathogenesy of PAM again. Thisstudy not only make up a domestic deficiency of this gene, but also establish afoundation to identify the pathogenesy of PAM and others microlith disease;supply referred pathway to treat these disease and establish an importantesperiment foundation to approach the function of SLC34A2 and signaltransduction furthermorely.