calcitonin is a 32-residue peptide hormone produced by specialized c - parafollicular cells of the thyroid glands in mammals or by cells of the ultimobranchial glands in reptiles and fish .
the nucleotide sequence and translation of human calcitonin are shown in figure 1 [ 1 , 2 ] .
calcitonin plays an important role in regulating calcium and phosphorus metabolism , decreasing blood calcium concentrations and inhibiting bone resorption .
natural calcitonin and synthesized analog are widely used in clinical practice for the treatment of postmenopausal osteoporosis , paget 's disease of bone , bone pain , spinal stenosis , acute pancreatitis , and gastric ulcer [ 3 , 4 ] .            following the increase of the proportion of the elderly people in the world ,
osteoporosis has become a major threat to the public health due to its high morbidity and mortality [ 59 ] .
low bone mass and deterioration of bone microarchitecture are the major characteristics of osteoporosis , which results in increased bone brittleness and thus is associated with an increased risk for fracture .
calcitonin is one of the effective and safe agents for the treatment of osteoporosis , along with its antalgic effect [ 3 , 4 ] .
calcitonins that are used in clinical practice are mainly extracted from the gills of salmon and pig thyroid glands .
however , these heterologous products are short of resources and thus expensive . in addition , the heterologous calcitonins are weak antigens and antibody - related resistance occurs in 40%70% of patients .
reported that transient or persistent nausea was experienced in about one third of patients , with diarrhea , vomit and local pain at the injection site being other side effects .
in addition , currently recommended nasal administration of calcitonin only reaches low concentrations in blood , but may cause squamous metaplasia in the nasal mucous membrane , which greatly restricts its long - term application .        
therefore , gene engineering techniques with human calcitonin ( hct ) gene as the objective gene may provide solutions overcoming the above drawbacks seen in clinical practice . however , although there have been some studies on the synthesis of calcitonin [ 14 , 15 ] , there is little information in literature on its application in gene therapy . for this purpose
, we constructed a recombinant eukaryotic expression plasmid , pcdna3.0-ig-hct , which contains human calcitonin gene and murine ig-chain leader sequence .
the recombinant plasmid was transferred into cells of murine fibroblast cell line , nih3t3 , and the expression and secretion of recombinant hct in nih3t3 cells were observed in this study .
the sequences of a murine ig-chain leader sequence   and hct gene [ 1 , 2 ] were divided into six fragments and used as pcr primers and templates for cdna synthesis .
the primers were as follows : :   5-caagcttatggagacagacacactcctgctatgggtactg-3 , :   3-tgaggacgatacccatgacgacgagacccaaggtccaaggtgaccactgcgc-5 ,   :   5-caggttccactggtgacgcggcccagccggccaggcgcgccgtacgggatccgtg-3 ,   :   3-gcgcggcatgccctaggcacgccattagactcatgaacgtacgacccgtgtatg-5 ,   :   5-cttgcatgctgggcacatacacgcaggacttcaacaagtttcacac gttccccca-3 ,   : 3-gttcaaagtgtgcaagggggtttgacgttaaccccaacctcgtggaattagatctg-5 ,   the sequence to be synthesized was shown in figure 1(a ) .
a dynamic template pcr technique was used to synthesize ig-hct ( figure 1(a ) ) [ 17 , 18 ] .
the pcr program included predenaturation at 95c for 5 minutes , 30 amplification cycles each consisting of denaturation at 94c for 30 seconds , annealing at 55c for 60 seconds , and extension at 72c for 50 seconds ; followed by further extension at 72c for 5 minutes .
the cloned cdna fragments were ligated into the pmd 18-t vector ( promega , usa ) to form pmd 18-t - ig-hct .
the recombinant vector pmd18t - ig-hct was amplified in   e. coli   jm109 and then extracted by a dna purification system ( promega ) according to the manufacturer 's instructions .
pmd18t - ig-hct was digested by   hindiii and xbai and then evaluated by 1.5% agarose gel electrophoresis .
the sequence of the recombinant ig-hct eukaryotic expression vector identified by the restriction analysis was determined by using fluorescent dideoxynuleotides on an automated dna sequencer ( gene core technologies co. ltd , shanghai , china ) .
the procedure of construction of the recombinant pcdna3.0-ig-hct eukaryotic expression vector was shown in figure 1(b ) . the cdna fragment of ig-hct from pmd 18-t vector was further subcloned into the pcdna3.0 ( promega ) to form a eukaryotic expression vector , pcdna3.0-ig-hct .
the recombinant expression vector pcdna3.0-ig-hct was amplified in e. coli   jm 109 and then extracted by a dna purification system ( promega ) .
the pcdna3.0-ig-hct retrieved from the gel was digested by   hindiii and xbai as previously described .
nih3t3 ( atcc ) cells were transfected with pcdna3.0-ig-hct by liposome , lipofectamine 2000 ( invitrogen , usa ) .
after 6-hours exposure , the normal culture medium , dulbecco 's modified eagle media ( dmem ) , supplanted with 10% fetal bovine serum ( gibco , usa ) was added into cells .
forty eight hours later , cells were exposed to 200 , 400 , 600 , 800 , 900 , and 1000 g / ml g418 ( gibco , usa ) , respectively , with 3 days for each concentration .
after 18 days of selection , g418-resistant clones were randomly picked up , and cultured in the medium containing 800 g / ml g418 .
single clones were transferred into and cultured in the medium with g418 concentration being reduced to 0.5 g / ml .
nih3t3 cells that were transfected with pcdna3.0 were used as controls . to demonstrate the mrna expression of ig-hct mediated by pcdna3.0-igk - hct vector in nih3t3 cells ,
first strand cdna was performed by an amv transcriptase kit ( promega ) according to the manufacturer 's instructions .
the sequences of primers used for the detection of ig-hct were 5-taatacgactcactatagggaga-3 ( 5-sense ) and 5-actcgtatcatccthccgaa-3 ( 3-antisense ) with a product of 212 bp .
for the detection of -actin mrna , we use a 5-sense primer ( 5-gggaaatcgtgcgtgacat-3 ) and a 3-antisense primer ( 5-tcaggaggagcaatgatcttg-3 ) , with a product of 385 bp .
pcr program was predenaturation at 95c for 5 minutes , and then 30 cycles each consisting of denaturation at 95c for 50 seconds , annealing at 55c for 50 seconds , extension at 72c for 50 seconds , and an extension at 72c for 7 minutes .
pcdna3.0-igk - hct - transfected , nontransfect ed and pcdna3.0-transfected nih3t3 cells were cultured in 6-well plates ( 1  10/ml ) .
one slide was put in each well of six - well plates for 24 hours to grow nih3t3 cells .
then , immunohistochemistry was performed on ethyl alcohol / acetone - fixed slides by immunohistological kit for hct ( zhongshan golden bridge , beijing , china ) .
supernatant of cultured pcdna3.0-igk - hct - transfected nih3t3 cells were subjected to western blotting after 10 fold condensation by lyophilization , with pcdna3.0-transfected nih3t3 cell culture enriched supernatant being used a control .
piscataway , usa ) , the blots were probed with the following antibodies : rabbit antihct ( chemicon international inc.,temecula , usa ) and goat antirabbit igg - hrp ( chemicon international inc . ) .
pcdna3.0-igk - hct - transfected , nontransfected and pcdna3.0-transfected nih3t3 cells were cultured in 6-well plates ( 1  10 cells / ml ) .
after the cells were cultured for 24 hours , secretion of hct in the supernatant was measured with a commercial hct detection kit ( cehmclin , beijing , china ) by using the radioimmuniassay method .
pcdna3.0-igk - hct - transfected , nontransfected and pcdna3.0-transfected nih3t3 cells were cultured in 6-well plates ( 1  10/ml ) for 24 hours.then , primarily isolated osteoclasts were settled onto the 6-well plates and cocultured with above nih3t3 cells , respectively , on 15 mm plastic coverslips which were placed on a metallic bracket in plates , and incubated for 7 days at 37c . as a positive control , salmon ct ( sct , novartis , basel , switzerland )
was added , at concentration of 1 ng / ml , into the coculture of nontransfected nih3t3 cells and osteoclasts . to determine the identity of osteoclasts
, cells were fixed and stained by using a tartrate - reisitant acid phosphatase ( trap ) stain kit ( sigma , usa ) according to the manufacturer 's protocol on day 7 of culture .
trap - positive multinucleated cells with more than three nuclei were considered to be osteoclasts ( figure 3 ) .
finally , the osteoclasts were observed under a converted microscope , and their numbers were counted and morphology observed .
database was set up with spss 10.0 software package ( spss inc , chicago , il ) for analysis .
the significance of differences was determined by anova and bonferroni 's modification of student 's   t - test . a difference with a p value of less than.05
the pcr product of pmd18t - ig-hct had the same length with designed ig-hct gene .
ig-hct - cdna was successfully inserted into pmd18 t as shown by hindiii and xbai digestion and electrophoresis ( data not shown ) .
it was demonstrated that the stop codon and endonuclease sites of ig-hct was successfully cloned into pmd18-t ( data not shown ) .
the ig-hct cdna was successfully ligated into pcdna3.0 by hindiii and xbai digestion and electrophoresis ( figure 1(c ) ) .
the 210 bp fragment corresponding to the ig-hct cdna amplified by rt - pcr was present only from pcdna3.0-igk - hct - transfected nih3t3 cells , but not from nontransfected or pcdna3.0-transfected cells ( figure 2(a ) ) .            
moreover , immunocytochemistry staining showed that expression of hct protein was detected in pcdna3.0-igk - hct - transfected nih3t3 cells ( figure 2(b)1 ) , but not in pcdna3.0-transfected or nontransfected cells ( figures 2(b)2 and 2(b)3 ) .
western blotting showed tha hct was positively stained in pcdna3.0-igk - hct - transfected cells , but not in pcdna3.0-transfected or nontransfected cell ( figure 2(c ) ) .          
in addition , radioimmunoassay displayed significantly higher secretion of hct protein in the culture supernatant ( 46  7.29 ng / ml ) of pcdna3.0-igk - hct - transfected nih3t3 cells than those of nontransfected cells ( 5.2  2.36 ng / ml ) and pcdna3.0-transfected cells ( 4.8  4.31 ng / ml ) ( both p < .05 ) .
trap staining showed that primarily isolated osteoclasts cocultured with nontransfected and pcdna3.0-transfected cells were multinucleate and spreading under a converted microscope ( figures 3(a ) and 3(b ) ) .
however , it was observed that osteoclasts became crumpled when cocultured with pcdna3.0-ig-hct - transfected nih3t3 cells and nontransfected nih3t3 cells plus sct group .
the number of trap - positive multicleate cells was significantly fewer when the primarily isolated osteoclasts were cocultured with pcdna3.0-ig-hct - transfected nih3t3 cells or with nontransfected nih3t3 cells plus sct , than with nontransfected or pcdna3.0-transfected cells ( table 1 , p < .01 ) .
osteoporosis is characterized with low bone mass and deterioration of bone microarchitecture which can cause decreased bone strength and an increased risk for fracture [ 59 ] .
calcitonin is one of the most effective reagents for osteoporosis with antalgic activities [ 3 , 4 ] .
it is believed that sct can inhibit bone resorption , reduce bone mass loss and relieve bone pain [ 20 , 21 ] .
but oral or nasal administration of calcitonin can cause many side effects in osteoporosis patients [ 12 , 13 ] .
otherwise , long - term application of animal calcitonins leads to a sharp activity decrease in clinical use of osteoporosis due to the accumulation of antibodies against these heterologous calcitonins .
therefore , gene therapy with calcitonin as an objective gene may represent an optimal resolution to overcome the above problems .         
hct can not be secreted after synthesized in the cytoplasm because the nucleotide sequence of hct1 - 32 lacks of a signal peptide , which greatly limits its application in gene therapy . in the present study
thus , hct can be secreted into the culture supernatant after it is synthesized in the cytoplasm . at the same time , the signal peptide is cleaved to avoid influencing the activities of hct . in this study ,
there are 19 or 20 complementary bases between p1 and p2 , p2 and p3 , p3   and p4 , p4 and p5 , and p5 and p6 . in the first stage of pcr , p1 and p2 ,
p3     and p4 , and p5 and p6 were used as primers and templates to amplify p1 - 2 , p3 - 4    and p5 - 6 . in the second stage ,
p1 - 2 , p3 - 4    and p5 - 6 were used as primers and templates to amplify p14    and p36 .
the sequence analysis and electrophoresis of pcdna3.0-ig-hct after digestion with hindiii and xbai demonstrated that the objective gene ig-hct was successfully cloned and ligated into the eukaryotic expression vector .
the 210 bp fragment corresponding to the ig-hct cdna was amplified by rt - pcr , and was expressed only from pcdna3.0-igk - hct - transfected nih3t3 cells .
the expression and secretion of hct were also detected in pcdna3.0-ig-hct - transfected nih3t3 cells by the immunohistological method , western blotting and the radioimmunoassay method .
these observations suggest that the target fragment was successfully integrated into the genome of infected nih3t3 cells , which was expressed successfully .        
it has been shown that calcitonin inhibits bone resorption and induces osteoclast apoptosis by binding to calcitonin receptors on osteoclasts .
we observed that primarily isolated osteoclasts cocultured with the supernatant of pcdna3.0-igk - hct - transfected nih3t3 cells became crumpled and their number was decreased compared with those cocultured in nontransfected and pcdna3.0-transfected nih3t3 cells , which suggests its profound bioactivities such as inhibition of the growth and alteration of morphology of osteoclasts .         
many in vivo active peptides are posttranslationally amidated at the c terminus , which is of great importance to their activities and stability .
eukaryotes can be used to make some modifications to products , which make it possible to directly produce amidated calcitonin . in our future research
, we will aim to couple express a rat amidating enzyme and hct - gly in nih3t3 cells , to obtain amidated bioactive hct .          
microencapsulation is a technique that encapsules an implant into a biocompatible selective membrane made from polymers , which can insulate the implant and the immune system of the recipient , but allow small molecules , electrolytes , oxygen , and metabolites to get through the membrane .
indeed , microencapsulated transplantation of cells and tissues has been demonstrated to be effective in the treatment of many diseases .
we propose that cell lines secreting hct encapsulated by alginate - polylysine - alginate membranes microcapsules and transplanted would constantly secret bioactive hct for a long perioid of time in vivo , and thus represent a new strategy for the treatment of osteoporosis .         in conclusion
the hct gene was effectively expressed in nih3t3 cells and secreted into the culture supernatant with the help of the murine ig-chain leader sequence .
this study provides experimental data for potential gene therapeutic usage of the recombinant hct for osteoporosis .
 
 aim . to construct a recombinant eukaryotic expression plasmid containing human calcitonin ( hct ) gene and express the gene in murine fibroblast nih3t3 cells . 
 materials and methods . a murine ig-chain leader sequence and hct gene were synthesized and cloned into pcdna3.0 to form the pcdna3.0-ig-hct eukaryotic expression vector , which was transfected into nih3t3 cells . 
 the mrna and protein expressions and secretion of hct were detected . 
 primarily cultured osteoclasts were incubated with the supernatant of pcdna3.0-igk - hct - transfected nih3t3 cells , and their numbers were counted and morphology observed . 
 results . 
 the expression and secretion of hct were successfully detected in pcdna3.0-igk - hct - transfected nih3t3 cells . 
 the number of osteoclasts was decreased and the cells became crumpled when they were incubated with the supernatant of pcdna3.0-igk - hct - transfected nih3t3 cells . 
 
 conclusion . 
 a recombinant eukaryotic expression vector containing hct gene was successfully constructed and expressed in nih3t3 cells . 
 the secreted recombinant hct inhibited the growth and morphology of osteoclasts . 
1. Introduction
2. Methods
3. Results
4. Discussion