Journal of Huazhoag University of Science and Technology [Med Sci]

144

25 (2).. 144-146, 2005

Immortalized Rat Astrocyte Strain Genetically Modified by Rat Preprogalanin Gene* AN Ke ( ~

-N), TIAN Yuke (~--~.ff4), YANG Hui (]@

~),

GAO Feng (r~

~r

WANG Peng (

Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

Summary: To construct an immortalized rat astrocyte strain genetically modified by rat preprogalanin gene (IAST/GAL) and detect its galanin (GAL) expression and secretion, a cDNA fragment of rat GAL in plasmid of pBS KS( + ) - G A L was inserted into eukaryotic expression vector pcDNA3.1 ( + ) by DNA recombinant technology, then the restriction enzyme digestion and DNA sequencing were carried out to evaluate the recombinant. The peDNA3.1 ( + ) - G A L and pcDNA3.1 ( + ) construct were transfected into immortalized rat astrocyte strain (IAST) by lipofectamine and the population of cells which stably integrated the construct was selected with 600 ~g/mL G418. Individual clones were screened and expanded into clonal cell strains. Detection of Neo gene was used to validate the success of the transfection. Immunocytochemical staining, RT-PCR and radioimmunoassay were used to detect the expression and secretion level of GAL. The recombinant had been successfully constructed by restriction enzyme digestion and DNA sequencing. Detection of Neo gene showed that the pcDNA3.1 ( + ) - G A L and pcDNA3.1 ( + ) have been successfully transfected into IAST. After selection by using G418, IAST/GAL and IAST/Neo cell strains were obtained.

IAST/GAL, IAST/Neo and IAST were immunostained positively for GAL, but the GAL average optical density of IAST/GAL was significantly higher than that of IAST/Neo and IAST ( P 0.01). The level of GAL mRNA expression and the supernatant concentration of GAL in cultured IAST/GAL were significantly higher than those of IAST and IAST/Neo ( P < 0 . 0 1 ) , but no significant differences were found between the IAST and IAST/Neo ( P > 0 . 0 5 ) . It was concluded that IAST/GAL strain was constructed successfully and it might provide a basis for the further study of pain therapy. Key words: preprogalanin; astrocyte; immortalized ~ cell strain; biologic minipumps

Galanin is a neuropeptide consisting of 29 or 30 (in humans) amino acids which is widely distributed in the central and peripheral nerve system and is involved in a variety of physiological and pathophysiological activities, including pain signaling. It has been proposed to play a central role in the inhibition of neuropathic pain. More recently, the strategy of transgenic cell grafts as "biological minipumps" to deliver anti-nociceptive molecules near the pain processing centers of the spinal cord represents a new frontier in the relief of neuropathic pain. T h e r e f o r e , in the present study, an immortalized rat astrocyte strain genetically modified by rat preprogalanin gene is constructed to provide a basis for the further study of transgenic cell transplantation for pain therapy. 1

MATERIALS AND METHODS

quence was kindly provided by Dr. David Wynick (Bristol Royal Infirmary, Bristol, E n g l a n d ) , and this clone containing the rat preprogalanin gene in pBS K S ( + ) has a unique Hind Ill/EcoR I site for the excision of a 521 bp rat galanin cDNA. 1 . 1 . 2 Reagents Lipofectamine TM 2000 and G418 were all purchased from Gibco B R L , USA. Restriction endonucleases ( H i n d Ill and EcoR I ) , T4 DNA ligase and R T - P C R kits were obtained from T a K a R a Biotechnology C o . , China. Rabbit anti-galanin polyclonal antibody was purchased from Chemicon, USA. Galanin radioimmunoassay kits was provided by the Department of Neurobiology of the Second Military Medical University, China. TRIzol RNA was from Shanghai H u a s h u n Co. , Ltd, China. PCR Primers were synthesized by Shanghai Bioasia Biotechnology Company, China. 1.2

1.1 ~ Materials 1.1.1 Cell Strain, Bacterium and Plasmids

Immortalized rat astrocyte strain ( I A S T ) Ell and E. coli strain DH5a were established and conserved by our laboratory. Plasmid pcDNA3. 1 ( + ) was purchased from Invitrogen, USA. The pBS K S ( + ) G A L construct encoding the rat preprogalanin seAN Ke, male, born in 1969, M. D. , Ph.D. " This project was supported by a grant from the National Natural Science Foundation of China (No. 30170905).

Methods

1 . 2 . 1 Construetion of Eukaryotie Expression Vector p c D N A 3 . 1 ( + ) - G A L Plasmid pcDNA3.1 ( + ) and pBS K S ( + ) - G A L were all digested with Hind HI and EcoR I , and the digested fragments were re-collected from the low melt agarose gel and fast ligated directly by T4 DNA ligase. T h e reaction product was transformed into E. coli ( D H 5 a ) , and the positive bacterial colony was selected. T h e recombinant pcDNA3. 1 ( + ) - G A L was identified by using restriction endonucleases digestive reaction and DNA sequencing.

AN Ke et al. Astrocyte Strain GeneticallyModified by Preprogalanin Gene 1.2. 2 Transfection of pcDNA3. 1 ( -4- ) and pcDNA3. 1 ( + ) - G A L into lAST IAST from log growth phase was transfected with pcDNA3. 1 ( + ) - G A L and peDNA3. 1 ( + ) by lipofectamine respectively. After selection with 600 g g / m L G418 for two weeks, the positive cell clone was picked and expanded into elonal cell strain. 1 . 2 . 3 Detection of Neo Gene in Positive Clones Both recombinant pcDNA3. 1 ( + ) - G A L and plasmid pcDNA3. 1( + ) have Neo marker gene. In order to find out whether gene transfection was successful, the expression of Neo gene in trasnfected clones was detected by RT-PCR. Isolation of total RNA from the positive cells and RT were performed with the commercially available TRIzol RNA and RT kits. The 237 bp Neo eDNA fragment was PCR- amplified using up-stream primer (5'- AGAGCA2TATTCTGCTATGAC -39 and downstream primer (5"-GCTTCAGTGACAACGTCGAG-3"). 5/IL of PCR products was run on 1.2 ~ agarose gel for electrophoresis and observed under ultraviolet light. 1.2.4 RT-PCR Analysis of GAL mRNA Expression Total RNA of IAST and positive elonal cells were isolated and reserve-transcripted as described above. PCR amplification of GAL and 13-actin eDNA fragment was performed with the following primer pairs: GAL: 5 "-GATCATTTAGCGACAAGCA-3' and 5"-TAGGTCTTCTGAGGAC~TG-3" (209 bp), 13-actin: 5"-ATGTTTGAGACCTTCAACAC-3 " and 5 "-CACGTCACACTTCATGATGG -3" (489 bp). ~-actin was used as internal control. The multiplex PCRs were performed in 50 btL reaction volume. PCR conditions consisted of 0. 2 t~g of cDNA 2/~L, 5 ~L 10XPCR buffer, 25 mmol/L MgC12 2/~L, 10 mmol/L dNTP Mix 1 ttL, 5 units of Taq DNA polymerase 1 gL and 10 gmol/L each pairs of target primers. Water was added to a final 50/~L reaction volumes. PCR was performed according to the following program: pre-denaturation at 94 C for 5 rain, followed by 35 cycles of 94 ~ for 30 s, 54 ~ for 30 s and 72 ~ for 1 rain, then a finial elongation step at 72 ~ for 10 rain. All PCR products were analyzed by using 1.5 agarose gel electrophoresis with ethidium bromide and photographed. The results were evaluated by MGIAS1000 analyst software (Bio-Rad Co. , USA). Level of GAL mRNA expression was presented as the ratio of the integral absorbency (IA) of the gene product compared with that of 13-aetin. 1 . 2 . 5 Immuncytochemistry for Detection of GALMethods for staining cell cultures were referred to the instruction of StreptAvidin Biotin Complex kit (Beijing Zhongshan Co. ,China). Transfected cells were cultured on slides and fixed in methanol for 15 min. After blocked with normal goat serum for 10 min at room temperature, the slides were incubated with rabbit anti-galanin polyclonal antibody (1 : 1000 dilution) for 48 h at 4 ~ and then with an anti-rabbit peroxidase for 30 min at 37 ~ The reaction was developed in the presence of DAB substrate. After reactions were completed, slides were covered and observed under microscope. The

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level of galanin expression was presented as the value of AOD (average optical density) which was calculated by the Microimage Analysis Software, (Olympus Optical Co. , Japan). 1. 2. 6 Radioimmunoassay for GAL Secretion in Cell Culture Medium After cultured for 5 days, the culture medium (each 3 mL) was collected and boiled in boiling water for 5 rain, and then it was centrifuged at 1000 r/rain for 10 rain at 4 ~ The secretion level of galanin in supernatant was determined by the ordinal and saturated procedure of radioimmunoassay (RIA) to add the preparations according to the instruction of kits. 1.3 Statistical Analysis Results were presented as ~ 4-s. The data were analyzed by one-way ANOVA . Comparison of differences between multiple individual means was made by employing the Tukey's honestly significant differences (HSD) method or the least-significant difference (LSD) test. A P value less than 0.05 was considered statistically significant. All of the analyses were performed by using SPSS (10.0) software package (SPSS Inc. , USA). 2

RESULTS

2.1 Identification of the Recombinant pcDNA3.1 ( + )-GAL Correct orientation of ligations was determined by restriction enzyme analysis. The recombinant pcDNA3. 1 ( + ) - G A L was digested with Hind HI and EcoR I . There were two predicted DNA bands of 5.4 kb of pcDNA3. 1 ( + ) and 521 bp of GAL fragment, the latter being as long as the short DNA band of plasmid pBS KS( -4- )-GAL by the same digestive endonucleases. The recombinant plasmid could be digested by Hind m or EcoR I , but not by BamH I. DNA sequencing and BLAST showed that the 521 bp nucleotides were identical to the GAL gene of the Genbank (100 %). 2.2 Identification of Positive Clones The results showed that the size of amplification product of Neo gene was identical to the expected one (237 bp), but there was no RT-PCR product from non-transfected cells (IAST). It was confirmed that both pcDNA3.1 (A-)-GAL and pcDNA3. 1 ( + ) were transfected into the IAST successfully, and they were named I A S T / G A L and IAST/Neo cell strain respectively. 2.3 Analysis of GAL mRNA Expression Comparison of the ratio of integral absorbency (IA G A L / I A 13-actin) among cells showed that there was no significant difference between IAST/ Neo and IAST (0. 5481_--4-0.022 vs 0. 58314-0. 038) (P > 0. 05), but both of them had significant differences as compared with that of I A S T / G A L (2. 641-+-0. 066, P < 0 . 01), suggesting that the expression of GAL mRNA was increased in those cells transfected with p c D N A 3 . 1 ( + ) - G A L .

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Journal of Huazhong University of Science and Technology [Med Sci] 25 (2): 144-146, 2005

2. 4 Immunocytochemistry for Detection of GAL Expression

Immunoeytochemical assay with anti-galanin antibody showed that G A L staining ( b r o w n ) was localized positively in the cytoplasm of all &~ee

Fig. 1

2.5

Immunocytochemical staining of GAL in the cultured cells (SABC X 1000) A:Immunocytochemical light staining of GAL in IAST, showed that expression of GAL was weak and localized primarily in cytoplasm. (DAB • 200) B:Immunocytochemical light staining of GAL in IAST/Neo, showed that expression of GAL was weak and localized primarily in cytoplasm. (DAB • 200) C:Immunocytochemical light staining of GAL in IAST/GAL, showed that expression of GAL was strong and localized primarily in cytoplasm. (DAB)< 200)

Galanin Secretion Level in Cell Culture Medium

The results are given in table 1. Table 1

The concentration of galanin in supernatant of cell culture medium ( x + s , ng/mL)

Groups

77

Galanin

lAST

10

0. 1198+0. 0154

IAST/Neo

10

0. 1219•

0146

IAST/GAL

15

2. 6738•

3347 ~

P~0.01 as compared with other two groups

3

ceils (fig. 1). T h e AOD value of G A L in I A S T / G A L (0. 7904 + 0. 0778) was significantly higher than those of the others ( P % 0 . 0 1 ) . No significant difference was found between I A S T / N e o and l A S T (0. 19434-0. 0115 vs 0. 2017-4-0. 0128, P > 0 . 0 5 ) .

DISCUSSION

T h e neuropeptide galanin, a 29-amino-acid peptide with C-terminal amidation, was originally isolated from porcine intestine by T a m e m o t o at the Karolinska Institute in Sweden c2]. It is widely distributed in the central and peripheral nervous systems, expression of the peptide is also detected in dorsal root ganglia ( D R G ) and spinal dorsal horn interneurones :a? . Recently, electrophysiological studies on animals with axotomized, chronic constriction injury and spinal nerve ligation have shown that intrathecal administration of galanin reduces the excitability of spinal cord. Moreover, intratheeal administration of the galanin antagonist M35 significantly potentiates the frequency and severity of axotomyinduced autotomy behavior E4]. Galanin often coexists with calcitonin gene-related peptide ( C G R P ) , substance P and enkephalin in primary afferents, and inhibits the input of nociception stimulation at the postsynaptic sites [~?. It is discovered that galanin receptor ( G A L R ) belongs to the G-protein coupled receptor family. Binding of galanin to

GALR1 has been shown to inhibit adenylyl cyclase and stimulate cAMP synthesis, neuronal hyperpolarization as a result of opening of A T P - d e p e n d e n t K + channels or direct closure of L-type Ca 2+ channels, as well as inhibition of production of inositol phosphate ~]. Moreover, on allodynic and non-allodynic Bennett rat models, a dose-dependent increase in mechanical threshold was seen in the allodynic group after injection of galanin and galanin receptor agonist ARM961, which binds with equal affinity to GALR1 and G A L R 2 , but no effect was observed in the GALR2 agonist ARM1896-treated group. In non-allodynic rats neither of the two galanin receptor agonists caused significant changes of mechanical threshold ET~. Although the role of galanin in pain signaling is complicated, the general consensus is that this peptide plays a predominantly inhibitory role in spinal cord transmission and acts to reduce neuropathic pain behavior Ea?. T r e a t m e n t of sensory neuropathies that result in chronic pain is one of the most difficult problems in modern clinical practice. T h e current treatments, such as traditional pharmacological approaches, although often effective for limited periods, induce tolerance and unacceptable systemic side effects. H o w e v e r , in the later of 1980s', transplants of primary adrenal medullary cells that release peptides and neurotransmitters offered a new direction in the treatment of chronic pain :97. N o w , with the development of molecular biology and genetic engineering, cell transplantation, using immortalized cell strains genetically modified to release anti-nociceptive molecules as " biological m i n i p u m p s ' , offers a reasonable and physiologic alternative and can function permanently and safely. This approach should be able to reduce or elim(Continued on page 197)

LV Liqun et a l . Effect of Metformin plus CPA on Non-obese PCOS Endocrinol Metab, 1997, 82:4075 Morin-Papunen L C, Vauhkonen L, Koivunen R Met al. Endocrine and metabolic effects of metformin versus ethinyl estradiol-cyproterone acetate in obese women with polycystic ovary syndrome: a randomized study. J Clin Endocrind Metab, 2000,85 : 3161 13 Attia G R, Rainey W E, Carr B R. Metformin directly in12

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hibits androgen production in human thecal cells. Fertil Steril, 2001,76:517 14 Dahlgren E, Landin K, Krotkiewski Met al. Effects of two antiandrogen treatments on hirsutism and insulin sensitivity in women with polyeystie ovary syndrome. Hum Reprod, 1998,13:2706 (Received Sept. 20, 2004)

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inate side effects associated with large doses of p h a r m a c o l o g i c a l agents required for centrally-acting pain-reducing agents El~ A s t r o c y t e s are the m o s t a b u n d a n t cells in the central n e r v o u s system. T h e y f o r m a s t r u c t u r a l and functional interface n e t w o r k b e t w e e n nonn e r v o u s tissues and n e u r o n s , w h e r e t h e y regulate the n e u r o t r a n s m i t t e r release and the synaptic inform a t i o n processing in the CNS. T h e y posses b e t t e r h i s t o c o m p a t i b i l i t y and lower i m m u n o g e n i c i t y w h e n implanted into the recipient's C N S , so it is a better choice of cell vehicle for transgenic cell t r a n s p l a n tation. In this s t u d y , an e u k a r y o t i c expression vector p c D N A 3 . 1 ( 4 - ) - G A L is c o n s t r u c t e d and t r a n s f e c t ed into I A S T i n v i t r o . A f t e r selection and identification, an immortalized rat a s t r o c y t e strain genetically modified by rat p r e p r o g a l a n i n gene ( I A S T / GAL) is c o n s t r u c t e d successfully. The RIA s h o w e d t h a t the c o n c e n t r a t i o n of galanin in the sup e r n a t a n t of I A S T / G A L culture m e d i u m was higher t h a n t h a t of I A S T / N e o and I A S T . T h i s result was also confirmed by i m m u n o c y t o c h e m i c a l staining for galanin and by R T - P C R of G A L m R N A . W e are led to conclude that I A S T / G A L could o v e r e x p r e s s and secrete higher level of rat galanin protein i n v i t r o . REFERENCES

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