(03257)Zhuo M et el.:Long Term PD Gene Therapy Using Immortalized Glial Cell

Long
Term Gene Therapy of Parkinson’s Disease Using Immortalized Rat Glial Cell Line
with Tyrosine Hydroxylase Gene

ZHUO
Ming^#, XU De-Hua^#, CAO Lei, XU Ling-Fei, YU Fu-Rong,
ZHENG Zhong-Cheng, LIU Xin-Yuan*

( Institute of Biochemistry and Cell
Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of
Sciences, Shanghai 200031, China )

Abstract        Glial
cell is an ideal vehicle for gene therapy of brain diseases. However, there are
many limits in using primary glial cells. Therefore, an immortalized rat glial
cell line (RGLT) was established by SV40 large T-antigen (LTag) gene from the
primary rat fetal glial cells. The RGLT cell was shown to be non-tumorigenic
after transplantation to nude mice (up to 4 weeks) and rat striatum (up to 18
months). Rat tyrosine hydroxylase (TH) gene was transfected into RGLT cell to
obtain RGLT-TH cell. The TH immunohistochemical staining and HPLC-ECD analysis
demonstrated the TH expression and dopamine(DA) production in RGLT-TH cells in
vitro. When implanting RGLT-TH cells into the striatum of 6-hydroxydopamine
(6-OHDA) lesioned hemiparkinsonism model rats, TH immunohistochemical staining
showed the TH presence in striatum and HPLC-ECD analysis held at 6 months after
cell implantation showed an increase of DA content in striatum. The asymmetric
rotation of rats receiving RGLT-TH cells was reduced by 50%－60% and this reduction persisted stably
at least for 18 months. These results suggest that the immortalized glial cell
line could serve as an ideal vehicle for therapeutic gene delivery system to
achieve a long-term gene therapy of neurodegenerative diseases.

(Abstract in Chinese)

Key
words     Parkinson’s disease; gene
therapy; glial cell; SV40 large T-antigen; tyrosine hydroxylase

Parkinson’s disease (PD) is a progressive
brain disorder that is characterized by the loss of dopaminergic neurons in the
substantia nigra pars compacta. As the current mainstay of treatment, the
dopamine (DA) precursor, L-3,4-dihydroxyphenylalanine (L-dopa), can effectively
control the symptoms at the beginning of the treatment. However, this efficacy
gradually weakened with time[1,2]. Recent studies suggest that L-dopa therapy
can be improved through continuously keeping the level of L-dopa in the central
nervous system (CNS) by repeated small dose of L-dopa[3,4]. Gene therapy, as an
effective strategy for continuous deliveries of therapeutic materials[5], is
thought to be an attractive way in PD therapy. TH, which catalyzes the
conversion of tyrosine to L-dopa, is the rate-limiting enzyme in the DA
biosynthesis. It has been widely used in the preclinical experiments of PD gene
therapy[6]. In our previous studies, we have investigated the therapeutic
effect of TH gene delivery in 6-OHDA lesioned PD rat model. Direct injection of
the lipofectin and plasmid DNA complex into the striatum of PD rats could
transfer TH gene into neurons and improve the PD behavior in a short time[7].
In order to achieve long-term transgene expression, we have transplanted TH
gene transfected myoblast cell to PD rats. The asymmetric rotation was
significantly reduced, and this reduction persisted for up to 13 months[8].
Even longer effect might be achieved if there is better vehicle.

Glial cell is a part of the neural tissue
and performs vital functions regarding the survival and maintenance of
neuron[9]. It is thought to be a promising vehicle for ex vivo gene therapy in
brain. However, it is difficult to obtain primary cell in large quantity and
the life span of primary cells in culture is limited as well. These restricted
the utilization of primary glial cells. Immortalization of primary glial cells could
dissolve this problem, which makes it possible for primary glial cells to serve
as the vehicle of therapeutic gene for gene therapy.

In present work, we established an
immortalized glial cell line (RGLT) by using SV40 large T-antigen (LTag). Then
TH gene was transfected into RGLT to obtain long-term TH expression and
behavior improvement in PD gene therapy on rat model.

1    Materials
and Methods

1.1   Plasmids

Full-length rat TH cDNA was obtained from
pUC6s-TH[7] by EcoRV and BglII site, and inserted into pIRES-puro (Clontech) by
EcoRV and BamHI site to obtain the plasmid pIRES-puro-TH. The control plasmid
pIRES-puro-EGFP was from Clontech.

1.2   Cell culture

Primary cultures of fetal glial cell from
Sprague-Dawley(SD) rat were prepared by the following procedure. Briefly,
brains from 14-d fetal rats were dissected under microscope. Target tissue was
isolated and kept in serum-free medium, then minced and filtered with a
400-hole filter. The filtered medium was centrifuged at 1000 r/min for 5 min
and the cell pellet was washed with serum-free medium for 3 times, then
resuspended in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL) supplied
with 10% (V/V) fetal bovine serum (Gibco BRL).

After 72 h culture, the plasmid pCMV-LTag
was transfected into the cells using LipofectAmine (Gibco BRL). Cells were then
selected by 0.6 g/L G418 (Gibco BRL) for 2 weeks to obtain single clone. When
stable clone obtained, glial fibrillary acidic protein (GFAP)
immunohistochemical staining was performed to confirm the phenotype of the
cells and LTag immunohistochemical staining was held to detect the expression
of LTag. This stable cell clone was named RGLT.

1.3   Immunohistochemical staining

Immunohistochemical staining was
performed as the following procedure. Briefly, samples were fixed in 4%
paraformaldehyde, then washed with PBS (pH 7.2) and quenched in methanol
containing 3% H₂O₂. After being washed with PBS, samples
were blocked in 10% normal horse serum (Gibco BRL) with 0.1% Triton X-100 in
PBS, followed by incubation with antibody overnight at 4 ℃. Samples were then washed with PBS and
incubated with biotin labeled IgG at 37 ℃
for 1 h. After being washed with PBS, samples were incubated in
streptavidin-biotin complex (Sino-American Biotechnology Company) in PBS at 37 ℃ for 1 h. After further being washed with
TBS (pH 7.4), samples were incubated with 0.05% 3,3′-diaminobenzidine, 0.01% H2O2 in TBS
until color developed, then washed 3 times with TBS and examined under
bright-field microscopy.

1.4   Tumorigenesis assay

Four nude mice received 1×107 RGLT cells each by subcutaneous
injection followed by an up to 4 weeks’ survey on tumor occurrence. In another
assay, 2×105 RGLT cells were
transplanted into striatum of four SD rats. The striatum was sectioned 2 weeks,
3 months or 1 year after transplantation to examine whether there is tumor
formation.

1.5   Lesions and behavioral testing

The hemiparkinsonism model rats were
prepared by injection of 6-OHDA to substantia nigra of SD rats [(220±10) g, male] as described previously[8].
After 2 weeks recover period, apomorphine(APO)-induced asymmetric rotation test
was performed to detect the extent of lesion. The test was sequentially
performed for 2 months. The rats showing consistently rotation of at least 7
rotations per minute for the interval 30 min were selected for cell
transplantation experiment.

1.6   Cell transplantation

Rats were randomized into RGLT-TH group
(n=20) or RGLT-EGFP group (n=15). 200 000 RGLT-TH cells were stereotactically
injected into 4 sites within the striatum of rats in RGLT-TH group as described
previously[8]. The rats in RGLT-EGFP group received 200 000 RGLT-EGFP cells.

1.7   HPLC-ECD analysis

Dopamine and 3,4-dihydroxyphenylacetic
acid (DOPAC) in cultured RGLT-TH cells or striatum extracts of rats were
measured by HPLC-ECD (200A HPLC system and ECD, BSA Inc). Rats were
anesthetized and killed by decapitation. Striatum of both sides was immediately
removed onto dry ice and stored at －70
℃ until further treatment. The
striatum was weighed and homogenized in 1 mL of ice-cold 0.05 μmol/L HClO4 for 30 s, followed by
centrifugation of 25 000 g at 4 ℃ for 30 min. Cultured cells in
monolayer were treated similarly. 20 μL
of the supernatant was injected into the column (octadecylsilane, ODS, 10 μm particles) with a mobile phase
consisting of 0.15 mol/L chloroacetic acid-NaOH buffer, 9.0 mmol/L
D-camphor-sulfonate (CSA), 10% methanol and 0.83 mmol/L EDTA, pH 3.2, 1.0
mL/min. Detection limit was 20 nA.

2    Results

2.1   Immortalization of primary fetal
glial cell

Primary glial cells from 14-d fetal SD
rat were transfected with the LTag expressing retroviral vector. Single clone
was obtained by screening with G418. GFAP immunohistochemical staining was held
to show cell type of the clone. The GFAP positive stain indicates the glial
cell type (data not shown). The expression of LTag in RGLT cell was detected by
LTag immunohistochemical staining. All of the cells showed LTag positive stain
[Fig.1(A)] while none of the primary glial cells were stained [Fig.1(B)].
In general, primary glial cells can passage only 10－20 generations in culture. This cell line
has now been passed over 50 generations, and shows the immortalization
property. It is named RGLT cell.

Fig.1       Immunohistochemical staining of
LTag on cultured cells

(A)
RGLT cells. (B) Primary glial cells. (C) Survival cells of isolated striatum
receiving RGLT cells transplantation and screened by 250 μg/mL
G418 for 2 weeks.

2.2   Transgene expression in RGLT cell

To investigate survival and transgene
expression in RGLT cell in vivo, 1×105
RGLT cells were transplanted into striatum of SD rat. The host striatum was
isolated 3 months after transplantation and cultured in vitro with screening by
250 mg/L G418 for 2 weeks. Survival cells showed positive stain in LTag
immunohistochemical staining [Fig.1(C)], suggesting that RGLT cell could
survive after transplantation into brain for a long time and maintain the
expression of transgene.

pIRES-puro-TH or pIRES-puro-EGFP was
transfected into RGLT cell using LipofectAmine respectively. Cells were
selected by puromycin (Gibco) at 6 μg/L
for 1 week. The stable clones were named RGLT-TH and RGLT-EGFP cell
respectively. TH immunohistochemical staining, as described previously [7], was
held to detect the expression of TH in RGLT-TH cells. The expression of EGFP in
RGLT-EGFP cells was confirmed using fluorescent microscope. The result showed that
all of the RGLT-TH cells were TH positive stain in culture [Fig.2(A)].
When cells were transplanted into the striatum of PD rats for 6 months, one rat
from RGLT-TH group and another from RGLT-EGFP group were killed. TH
immunohistochemical staining was held on the brain slice from rat received
RGLT-TH cells. The widely positive stain of TH indicated that RGLT-TH cells
could survive and express TH in rat brain as shown in Fig.2(B). It also
suggests that RGLT-TH cell could integrate within the host tissue after
transplantation, rather than gathering around the needle track. The section
from rat received RGLT-EGFP cells showed green fluoresce when emitted by
ultraviolet light [Fig.2(C)], suggesting a long time expressing of EGFP
in RGLT-EGFP cells after cell transplantation.

Fig.2       Expression of transgene in
RGLT-TH cell and RGLT-EGFP cell

(A) Immunohistochemical staining of TH on
cultured RGLT-TH cells. (B) Immunohistochemical staining of TH on the striatum
section of rat 6 months after RGLT-TH cell transplantation. (C) The striatum
section of rat receiving RGLT-EGFP cells under fluorescent microscope with
ultraviolet light 6 months after cell transplantation.

2.3   Tumorigenesis assay

Subcutaneous injection of 1×10⁷ RGLT cells into nude mice
had not caused any tumor occurrence in a 4 weeks’ survey. Transplantation of 2×10⁵ RGLT cells into rat striatum
had not caused any tumor occurrence in a survey as long as 1 year. The
photographs of brain slice at 6 months after cell transplantation showed that
there were no tumors in the brain of rats received either RGLT-TH cell [Fig.2(B)]
or RGLT-EGFP cell [Fig.2(C)]. Moreover, no tumors were found in all rats
transplanted with RGLT-TH cells or RGLT-EGFP cells in our 18 months survey. All
these data suggests that RGLT cells have no tumorigenic effect in vivo.

2.4   HPLC-ECD analysis

Cell extracts from both RGLT-TH cells or
RGLT-EGFP cells were detected by HPLC-ECD. The results showed there were (59.5±4.6) ng DA and (0.53±0.08) ng DOPAC per 106 RGLT-TH cells, but
none of them were found in RGLT-EGFP (data are 6 months after cell transplantation, DA
and DOPAC in striatum from RGLT-TH group and RGLT-EGFP group were measured by
HPLC-ECD. Results are shown in Table 1. As the results show, lesion of
6-OHDA significantly decreases the DA content in rat striatum. However,
transplantation of RGLT-TH cells increases the contents of DA and DOPAC in
striatum to even higher than nondenervated striatum, indicating the strong
ability of RGLT-TH cell in DA synthesis in rat striatum. 18 months after cell
transplantation, the contents of DA and DOPAC in RGLT-TH group were still five
times to the RGLT-EGFP group (Table 2) showed that RGLT-TH cells still
had the ability in increase the DA content.

Table 1   Results of HPLC-ECD in the extract of striatum at 6
month after cell transplantation

The striatum of RGLT-TH and RGLT-EGFP
group is from denervated striatum with cell transplantation and the
nondenervated group is from the opposite striatum of the same rat from RGLT-TH
group. Data are represented as

Table 2   Results of HPLC-ECD in the extract of striatum at 18
month after cell transplantation

Tissues
are from denervated striatum of RGLT-TH or RGLT-EGFP group. Data are
represented as

2.5   Behavior testing

Eight rats were picked up randomly from
RGLT-TH group and eight from RGLT-EGFP group for the behavior testing. The
APO-induced rotation was measured every 4 weeks. As shown in Fig.3, rats
from RGLT-EGFP group kept their original asymmetric rotational behavior.
However, all the rats from RGLT-TH group had their rotation behavior reduced.
The reduction was more than 50% and persisted stably up to 18 months. This
result indicates using RGLT-TH cells in cell transplantation therapy could gain
significant, stable and long-time therapeutic effect.

Fig.3       Apomorphine-induced asymmetric
rotational behavior on PD rats

The
asymmetric rotation was measured every 4 weeks and represented as percentage of
the original rotation speed before cell transplantation. Data are x±s
values of 8 animals.

3    Discussion

In this study, we established an
immortalized glial cell line RGLT by using SV40 large T-antigen. This cell line
could express DA after transfection of TH gene. When RGLT-TH cells were transplanted
into striatum of PD rats, the DA content was significantly increased in rat
striatum and the asymmetric behavior was dramatically decreased. The
therapeutic effect stably persisted at least 18 months till the end of the
study.

In general, immortalized cell lines are
easy to manipulate and can be produced in unlimited numbers in culture.
Therefore, they have been used in first attempt in ex vivo gene therapy.
However, malignant transformation may occur during the immortalization
procedures and carry the risk of tumorigenesis. Previous studies have found
that brain contains inhibitory factors specific to the LTag[10]. The
expressions of LTag gene, as well as the proliferation of LTag-immortalized
cells, were inhibited following transplantation in brain[11]. These results
make it possible to the usage of LTag immortalized cell line in brain with
enough safety. In our work, we have held strict observation on tumorigenesis of
RGLT cells in vivo. The result showed RGLT cell could not form tumor both in
subcutaneous of nude mice and rat striatum. Even at 18th month after cell
transplantation, when the striatum were isolated form rat brain, there was
still no tumor found. These results suggest that RGLT cell do not have
tumorigenic effect. Besides the observation of tumorigenesis, we have also
observed whether RGLT cells could cause inflammatory response after cell
transplantation. Moreover, evidence of inflammatory response could not be
observed in all the rat brains received ether RGLT-EGFP cells or RGLT-TH cells
without immunosuppression. These data suggest that RGLT is neither tumorigenic
nor immunogenic in rat brain. It may serve as an unlimited, ready-to-use source
of nonautologous cell for transplant therapy.

Besides safety, efficacy is also important
in gene therapy. As TH being the rate-limiting enzyme, TH-only gene therapy is
effective at the beginning. However, the biosynthesis of DA includes at least 3
enzymes: TH, GTP cyclohydrolase (GCH) and aromatic-L-amino-acid decarboxylase
(AADC)[12,13]. As the expression of both AADC and GCH has been shown to greatly
decrease in lesioned brain[13,14], the ability of converting L-dopa into DA of
neurons decreases with time. It was reported that in gene therapy using TH and
AADC genes only, the efficacy is not as strong as that using all the three
genes[15]. It needs the expression of all these three genes to make a
therapeutic effect by the synthesis of DA in most cells, but it is hard to
establish a stable cell line with three genes transferred and make the genes
express simultaneously. Since it has been reported that glial cell can produce
DA from L-dopa[16], gene transfer with TH gene alone could be sufficient
to make the synthesis of DA. This makes glial cell to be an ideal vehicle for
PD gene therapy. Moreover, the close apposition of glial cell to neurons and
their capability of secreting a wide variety of substance[17] increase
therapeutic efficacy of glial cell in delivering neuroactive molecules to
neurons. In our work, we have confirmed that RGLT-TH cells could synthesize DA
in vivo. 6 months after transplantation of RGLT-TH cells, the DA content in PD
rats’ striatum was dramatically increased to a level even higher than the
nondenervated site (Table 1). In coincident with the increase of DA, the
asymmetric rotational behavior of rats was also decreased to 50% (Fig.3),
which is similar to the effect of gene therapy using TH, AADC and GCH genes
together[15]. These results indicate RGLT-TH cell has strong capability in DA
synthesis and can effectively improve PD behavior.

Besides the ability of DA synthesis,
glial cell is a kind of long life cell and a part of the neural tissue. It is
an ultralente vehicle in brain. Glial cell also has stronger resistance to oxidative
damage, a byproduct of DA synthesis, than neuron[18]. RGLT-TH, as a derivative
of glial cell, gains these advantages. In previous work, using other cells in
PD gene therapy, the therapeutic effect could only maintain limited time. Rat
marrow stromal cells (rMSCs) could only maintain to be effective for 87 d[19].
Using baby hamster kidney (BHK) cell could keep transgene expressing for 6
months[20]. Fetal porcine ventral mesencephalon grafts have up to 10 months
therapeutic effect[21]. Effect of myoblasts could maintain up to 13 months in
our lab before[8]. While in our work using RGLT-TH cell, the improvement in
asymmetric rotation could stably keep as long as 18 months. Considering that
rats have very limited life and our rats is 23 months old at the end of our
experiment, keeping 18 months’ therapeutic effect is surely a great advance. If
using long life span model animal, we would surely achieve even longer
effective therapeutic time.

In contrast to 6th month after cell
transplantation, the DA and DOPAC content in rat striatum received RGLT-TH
cells decrease a lot at 18 month after cell transplantation. One reason maybe
the effect of aging, since the rats are 23 months old at that time, even normal
rat will decrease a lot in the DA content in striatum[22]. Another reason may
be the shut-off of the CMV promotor. Using glial cell specific promotor like
GFAP promotor maybe a way to resolve this problem. As the GFAP promotor is not
as strong as the CMV promotor, it may not cause a so much high level of DA at
the 6th month, but could maintain the DA level stably for a longer time.

Taken together, our work proved that
genetically modified glial cell, RGLT-TH cell, could survive in the host brain,
synthesize DA and decrease the asymmetrical rotational behavior in a long time
without the risk of tumorigenesis and immunogenicity. It advocates the
potential application of glial cells in gene therapy of neurodegenerative
diseases.

____________________________

Acknowledgements     We thank Dr. Ge Kai for the plasmid pCMV-LTag.

References

1     Duvoisin RC. Overview
of Parkinson’s disease. Ann NY Acad Sci, 1992, 648: 187－193

2     Raymon HK, Thode S,
Gage FH. Application of ex vivo gene therapy in the treatment of Parkinson’s
disease. Exp Neurol, 1997, 144(1): 82－91

3     Schuh LA, Bennett JP
Jr. Suppression of dyskinesias in advanced Parkinson’s disease. I. Continuous
intravenous levodopa shifts dose response for production of dyskinesias but not
for relief of parkinsonism in patients with advanced Parkinson’s disease.
Neurology, 1993, 43: 1545－1550

4     Obeso JA, Grandas F,
Herroero MT, Horowski R. The role of pulsatile versus continuous dopamine
receptor stimulation for functional recovery in Parkinson’s disease. Eur J
Neursci, 1994, 6: 889－897

5     Gage FH. Cell therapy.
Nature, 1998, 392(6679 Suppl): 18－24

6     Wolff JA, Fisher LJ, Xu
L, Jinnah HA, Langlais PJ, Iuvone PM, O’Malley KL et al. Grafting fibroblasts
genetically modified to produce L-dopa in a rat model of Parkinson disease.
Proc Natl Acad Sci USA, 1989, 86(22): 9011－9014

7     Cao L, Zheng ZC, Zhao
YC, Jiang ZH, Liu ZG, Chen SD, Zhou CF et al. Gene therapy of Parkinson disease
model rat by direct injection of plasmid DNA-lipofectin complex. Hum Gene Ther,
1995, 6: 1497–1501

8     Cao L, Zhao YC, Jiang
ZH, Xu DH, Liu ZG, Chen SD, Liu XY et al. Long-term phenotypic correction of
rodent hemiparkinsonism by gene therapy using genetically modified myoblasts.
Gene Ther, 2000, 7: 445－449

9     Bacci A, Verderio C,
Pravettoni E, Matteoli M. The role of glial cells in synaptic function. Philos
Trans R Soc Lond Biol Sci, 1995, 354(1381): 403－409

10    Clarkson ED, la Rosa FG,
Edwards-Prasad J, Kumar S, Kumar A, Cole W, Freed CR et al. Brain contains
inhibiting factors specific to the large T-antigen gene. Cancer Lett, 1998,
122(1-2): 31－36

11    la Rose FG, Adams FS, Krause
GE, Meyers AD, Edwards-Prasad J, Kumar R, Freed CR et al. Inhibition of
proliferation and expression of T antigen in SV40 larger T-antigen gene-induced
immortalized cell following transplantations. Cancer Lett, 1997, 113: 55－59

12    Levine RA, Miller LP,
Lovenberg W. Tetrahydrobiopterin in striatum: Localization in dopamine nerve
terminals and role in catecholamine synthesis. Science, 1981, 214(4523): 919－921

13    Melamed E, Hefti F, Pettibone
DJ, Liebman J, Wurtman RJ. Aromatic L-amino acid decarboxylase in rat corpus
striatum: Implications for action of L-dopa in parkinsonism. Neurology, 1981,
31(6): 651－655

14    Kaddis FG, Clarkson ED, Weber
MJ, Vandenbergh DJ, Donovan DM, Mallet J, Horellou P et al. Intrastriatal
grafting of Cos cells stably expressing human aromatic L-amino acid
decarboxylase: Neurochemical effects. J Neurochem, 1997, 68(4): 1520－1526

15    Shen Y, Muramatsu SI,
Ikeguchi K, Fujimoto KI, Fan DS, Ogawa M, Mizukami H et al. Triple transduction
with adeno-associated virus vectors expressing tyrosine hydroxylase,
aromatic-L-amino-acid decarboxylase, and GTP cyclohydrolase I for gene therapy
of Parkinson’s disease. Hum Gene Ther, 2000, 11(11): 1509－1519

16    Juorio AV, Li XM, Walz W,
Paterson IA. Decarboxylation of L-dopa by cultured mouse astrocytes. Brain Res,
1993, 626(1-2): 306－309

17    Tacconi MT. Neuronal death:
Is there a role for astrocytes? Neurochem Res, 1998, 23(5): 759－775

18    Muller DP. Neurological
disease. Adv Pharmacol, 1997, 38: 557－580

19    Schwarz EJ, Alexander GM,
Prockop DJ, Azizi SA. Multipotential marrow stromal cells transduced to produce
L-DOPA: Engraftment in a rat model of Parkinson disease. Hum Gene Ther, 1999,
10(15): 2539－2549

20    Shingo T, Date I, Yoshida H,
Ohmoto T. Neuroprotective and restorative effects of intrastriatal grafting of
encapsulated GDNF-producing cells in a rat model of Parkinson’s disease. J
Neurosci Res, 2002, 69(6): 946－954

21    HogenEsch RI, Koopmans J,
Copray JC, van Roon WM, Kema I, Molenaar G, Go KG et al. Fetal porcine ventral
mesencephalon graft. Determination of the optimal gestational age for
implantation in Parkinsonian patients. Exp Brain Res, 2000, 132(3): 345－350

22    ThyagaRajan S, MohanKumar PS,
Quadri SK. Cyclic changes in the release of norepinephrine and dopamine in the medial
basal hypothalamus: Effects of aging. Brain Res, 1995, 689(1): 122－128

_______________________________________

Received:
July 8, 2003          Accepted:
September 16, 2003

This
work was supported by a grant from the National High Technology Research and
Development Program of China (863 Program) (No. BH-03-04-01)

^#Who contributed equally to
this work

*Corresponding
author: Tel/Fax, 86-21-54921126; e-mail, [email protected]

Updated
at: 2003-12-16