Effects of pirenzepine ophthalmic solution on form-deprivation myopia in the guinea pigs

Background Nonselective muscarinic receptor antagonist, atropine, was believed to inhibit myopic progression. The purpose of this study was to determine the efficacy, through topical administration, of the M1-selective muscarinic antagonist pirenzepine in preventing experimentally induced form-deprivation myopia in guinea pigs.Methods Fifty-three guinea pigs, which underwent monocular deprivation with their eyelids sutured, were divided into 6 groups. Three groups were treated with 1%, 2% or 4% pirenzepine ophthalmic solutions; the fourth group with atropine; the fifth with saline and the last group left untreated. Ocular refraction, in vivo biometric measurements and wet eye weight were collected before and after the experiment. All the eyes were finally enucleated for histopathological examination to evaluate the possible toxic effects on ocular structures.Results Animals untreated or treated with saline produced (-2.31±1.47) D and (-2.25±0.88) D of axial myopia respectively. Those treated with 1% pirenzepine ophthalmic solution produced relative myopia of (-1.63±0.48) D, and those under the treatment of 2% and 4% pirenzepine ophthalmic solution only developed a relative myopia of (-0.89±0.42) D and (-0.70±0.41) D (F=9.56, P<0.05). The significant reduction in myopia in 2% and 4% pirenzepine treated animals was caused by significantly less vitreous chamber elongation and axial elongation of the deprived eyes ［2% group: (0.009±0.052) mm, 4% group: (0.006±0.078) mm］ when compared with untreated, saline treated or 1% pirenzepine treated guinea pigs ［(0.057±0.056) mm, (0.064±0.053) mm and (0.033±0.035) mm, respectively］. Histological examinations revealed no obviously toxic effects on the eyes treated with pirenzepine.Conclusion Topical administration of the M1-selective muscarinic antagonist, pirenzepine, can prevent induced form-deprivation myopia in guinea pigs by inhibiting axial elongation without obvious damage to ocular tissues.     

CLINICAL AND EXPERIMENTAL STUDIES OF LARGE AMPLITUDE ACTION POTENTIAL OF THE SUFFERED FACIAL MUSCLES IN INTRATEMPORAL FACIAL NERVE PARALYSIS

Objctive. To testify the phenomenon that large amplitude action potential appears at the early stage oil facial paralysis, and to search for the mechanism through clinical and experimental studies. Patients(aninmls) and methods. The action potentials of the orbicular ocular and oral museles were recorded in 34 normal persons by electromyogram instrtiments. The normal range of amplitude percentage was found out according to the normal distribution, One hundred patients with facial paralysis were also studied. The action potentials of facial muscles were recorded ia 17 guinea pigs before and after the facial nerve was comp~ and the facial nerve was examined under electromicroscope before and after the compression.Results. The amplitude percentage of the suffered side to the healthy side was more than 153 percent in 6 of the 100 patients. Large amplitude action potential occured in 35 per cent guinea pigs which were performed the experiment of facial nerve compression. Electromicroscopic examination revealed separation of the lammae of the facial nerve‘s myelin sheath in the guinea pigs which exhibited large amplitude action potential Conclusion. The facial nerve exhibited a temporary over-excitability at the early stage of facial nerve injury in scane patients and guinea pigs. If the injury was limited in the myelin sheath, the prognods was relatively good.     

The Effect of Ginkgo Biloba Extract on the Expression of PKCα in the Inflammatory Cells and the Level of IL-5 in Induced Sputum of Asthmatic Patients

To investigate the effect of the Ginkgo Biloba Extract （GBE） on the asthma and examine its possible mechanisms, 75 asthma patients were divided into 4 groups and the patients were respectively treated with fluticasone propionate for 2 weeks or 4 weeks, or treated with fluticasone propionate plus GBE for 2 weeks or 4 weeks. Fifteen healthy volunteers served as healthy controls. Sputum inhalation with inhaling hypertonic saline （4%-5%） was performed. Lung ventilatory function and forced expiratory volume in one second （FEVI） were measured. The numbers of different cells in induced sputum were calculated. The expression of PKCα in the cells was immunocytochemically detected and the percentages of positive cells in different cells were counted. Interleukin-5 （IL-5） in sputum supernatants was detected with enzyme-linked immunosorbent assay. The percentage of eosinophils, lymphocytes, PKCα positive inflammatory cells and the concentration of IL-5 in asthmatic patients were higher than those in the controls （P〈0.05）, and the eosinophils, lymphocytes, positive expression of PKCα and the level of IL-5 were significantly decreased in asthmatic patients after they were treated with fluticasone propionate or fluticasone propionate plus GBE. However, they were still significantly higher than those of the controls. Compared to the group treated with glucocorticosteroid for 2 weeks, no significant decrease was found in the percentage of eosinophils, lymphocytes, PKCα positive inflammatory cells and the IL-5 in the supernatant of induced sputum. Compared with the group treated with glucocorticosteroid for 2 or 4 weeks, significant decrease in the same parameters was observed in the group treated with fluticasone propionate and GBE for 4 weeks. The IL-5 level in the supernatant of induced sputum was positively correlated with the percentage of PKCα-positive inflammatory cells and the percentage of eosinophils in the induced sputum in asthma patient groups respectively （n=150, r=0.83, P〈0.01; n=150,     

Effect on cochlea function of guinea pig after controlled release recombinant human bone morphogenetic protein 2

Background The recombinant human bone morphogenetic protein 2 （rhBMP-2） has been used to induce osteogenesis in animals＇ middle ear and this technique is possible to be used to reconstruct the defects of ossicles. The side effects of the rhBMP-2 in middle ear should be observed before using in clinic. Thus we prepared the controlled release rhBMP-2 and implanted it into the acoustic bulla of guinea pigs. The effect on the cochlea was observed. Methods We prepared the acellular cancellous bone, accompanied with rhBMP-2. The material accompanied with rhBMP-2 was implanted into one acoustic bulla of the animal and the opposite side of the acoustic bulla was implanted with acellular cancellous bone without rhBMP-2. Totally 20 guinea pigs were undergone this procedure. After the operation, the auditory brainstem response （ABR） of the animals was tested according to the time sequence. Three months after the operation, the animals were sacrificed. The osteogenesis induced by rhBMP-2, the acoustic bulla and cochlea affected by rhBMP-2 were observed. The structures of hair cells were observed after silver nitrate staining. Results The animals were recovered soon after surgery. The hearing thresholds of the animals were declined slightly just after the surgery and come back completely after 3 months. Also, the bulla and cochlea were normal in shape. The osteogenesis occurred in the pore of the acellular cancellous bone with rhBMP-2. There was not any abnormal hyperplasia of bone in the bulla and cochlea. The articulation between the stapes and oval window was not merged. The shapes of the hair cells were normal and there was no obvious deletion of the hair cells compared with control group. Conclusions The controlled release rhBMP-2 transplanted into the middle ear could induce osteogenesis in the bulla of the animals. It did not affect the shape of the bulla and the hearing threshold of the animal, and did not induce the abnormal hyperplasia of bone in the bulla and might be used to reconstruct the defects of ossicles.     

Effect of noise on the auditory center

Adult guinea pigs with normal Preyer's reflexes were used in this experiment and were di-vided into 3 groups according to the intensity of noise and exposure time.Auditory brainstemevoked response was recorded before and after exposure to noise.Clicks were presented monaurallyat a rate of high (50Hz) as well as low (10Hz).The superior olive and inferior colliculus were ob-served under an electron microscope after the guinea pigs were exposured to 110dB noise for30min.The average shift of wave Ⅳ amplitude-intensity function curve was more than that ofwave Ⅰ after exposure to intensive noise.The difference value of wave \ latency from low(10Hz) to high (50Hz) stimulus click rate decreased and was negatively correlated with the exposuretime.Ⅰ-Ⅴ,Ⅲ-Ⅴ and Ⅳ-Ⅴ interpeak latencies were shorter after exposure to noise.Themitochondria and neurilemma swelling were found in the superior olive and inferior colliculus afterexposure,Some mechanisms of the changes of auditory center were discussed in this paper.     

Co-culture of Mouse Embryos with Oviductal Cells of Guinea Pigs and Mice

Oviductal cell cultures were prepared from mice or guineo pigs at different days of estrus eycte and polarized or unpolarized mouse oviductal cells on the development of 1-cell mouse embryos in co-culture system were investigated. One-cell mouse embryos in co-culture with mouse metestrus and guinea pig Day 1 after ovulation had better development of embryos ( 73% and 79% of morula blastocysts, respectively). Co-culture of embryos with oviductal cells was improved when progesterons was added to the medium. Supplementing the co-cutture media with 20 ng/ ml estradiol-17β decreased the percentage of embryos forming blastocyst. The results showed that there was a very tow species specificity for oviductat cells and guinea pig oviductal cells. This provides a good possibitity for development of mouse embryos. The results also showed that good development for the 1-cell mouse embros is not cell polarity dependent.     

The Influence of LHRH, EB and PGF2α on the Development of Ovarian Follicles in Guinea Pigs

Effects of luwinizing hormone releasing hormone ( LHRH), estradiot benzoate (EB), prostaglandin (PGF2α) and the age of animals on the development of ovarian follicles in immature and mature guinea pigs were studied. Administration of 5ng LHRH / hr, 10 times a day, starting from 20-30 days Of age induced the flrst vaginal opening within 4 days, and 5 of the 10 animals had first ovulation. Tire results also show that appropriate doses of EB facilitated the development of ovary. The granulosa cells of guinea pigs at different ages were cocuttured with the pituitart, glands of mature ones in vitro. Results indicate that the responsiveness of granulosa cells of 20-day-guinea prgs to gonadotropins was similar to that of the matured ones. Effects of gonadotrophin on ovulation in hysterectomized and PGF2α or saline-treated animals were tested. Results demonstrate that when PGF2α was given simultaneously with hCG or FSH, 80% of the guinea pigs ovulawd and had fresh, healthy looking corpus tuteum. It is concluded that increased LHRH release from hypothatamus may be the key factor for tire onset of puberty in guinea prgs. EB and PGF2α can influence the development of the ovary.     

Roles of Protein Kinase C and Fructose in Hepatic Injury Caused by Obstructive Jaundice

Summary： The regulating mechanism in hepatic injury caused by obstructive jaundice （OJ） was examined in this study. Rat hepatocytes were harvested by in situ collagenase perfusion and subjected to primary culture. The heptocytes were pre-treated with various concentrations of protein kinase C （PKC） agonist PMA and its inhibitor chelerythrine and cultured for 20 min. After the treatment, 50μmol/L glycochenodeoxycholate （GCDC） was added and the cells were cultured for an additional 24 h. Cells were then detected by flow cytometry （FCM） and TUNEL. After hepatocytes were treated with different concentrations of fructose and 100μM GCDC, the cells were examined by FCM and TUNEL. Experimental obstructive jaundice （BDL） was induced by double ligation of the bile duct. After BDL, the rats were fed with or without fructos and sacrificed 3, 7, 14 and 21 days after the ligation. The apoptotic status was observed in liver of all rats with TUNEL and PKC protein in liver of OJ was studied by immunohistochemical method. Our results showed that PMA increased GCDC-induced apoptosis and chelerythrine decreased GCSX-induced apoptosis in a concentration-dependent manner. After the treatment with fructose of different concentrations, 100μM GCDC decreased the apoptotic rate and the apoptotic rate decreased with the increase of fructose concentration. The apoptotic rate of liver was related to the time of OJ. Without the treatment of fructose, PKC and apoptosis index （AI） were highest 14 days after the bile duct ligation. With the treatment of fructose, apoptosis index （AI） and PKC were decreased from the 14th day after the bile duct ligation. It is concluded that PKC is involved in the regulation of apoptosis in the liver cells with OJ and plays important roles in the development and progression of liver injury caused by OJ. Fructose can protect hepatocytes in the bile salt-induced apoptosis by regulating PKC.     

The Relationship of the Expression of Estrogen Receptor in Cartilage Cell and Osteoarthritis Induced by Bilateral Ovariectomy in Guinea Pig

To investigate the estrogen receptor（ER） expression in cartilage cell in the development of oste0arthritis induced by bilateral ovariectomy in guinea pig and to find their relationship. 30 two-month-old female guinea pigs were randomly divided into two groups （n=15 each） ： sham operation （control）group and ovariectomized group （OVX）; Scanning electorne microscope （SEM） and transmission electron microscope （TEM） were obtained to analysis the cartilage degeneration of the hind limb knee joint after 6 and 12 weeks of ovariectomy. Dextran-Coated-Charcoal （DCC） was taken to quantitively detect the expression of ER. The serum levels of estrogen and gestone were detected by immune contest assay. The results showed that ER do exist in the cartilages of the guinea pigs, with higher expression in the control group than in OVX group at the same time point （P〈0. 05）. It was increased also at 12 th week after operation than that of preoperation. The blood serum levels of estrogen and gestone showed a similar tendency to the expression of ER. Joint cartilage degeneration detected by SEM and TEM could be found at 6 th week, but severe degenerative lesions at 12 th week in the OVX group compared with the control group （P〈0.01）. The data suggested that bilateral ovariectomy in guinea pig lead to severe os.teoarthritis which mighgt be related to the lower serum level of estrogen and the downregulation of the expression of ER in the cartilage also.     

Changes of CD4＋ CD25＋ Regulatory T Cells in Peripheral Blood in Patients with Hepatocellular Carcinoma Before and after TACE

This study investigated the changes of CD4 CD25 regulatory T cells (Tregs) in periph-eral blood of patients with hepatocellular carcinoma before and after transcatheter arterial chemoem-bolization (TACE). The proportion of CD4 CD25 Tregs among CD4 T lymphocytes in peripheral blood of 33 patients with hepatocellular carcinoma was determined by flow cytometry before, 1 week and 1 month after TACE. And 25 healthy volunteers served as control. One month after TACE, the patients were divided into two groups: 22 in group A, who were in stable condition or getting better; and 10 in group B, who were deteriorating. One patient died and was excluded. The results showed that the percentage of CD4 CD25 Tregs among CD4 T lymphocytes did not significantly change in the 33 patients 1 week after TACE as compared with that before TACE, however, the difference was significant (P<0.01) between the patients with hepatocellular carcinoma and the healthy subjects. The percentage of CD4 CD25 Tregs among CD4 T lymphocytes in group A 1 month after TACE was decreased significantly in comparison with that before and 1 week after TACE (P<0.01), whereas, that in group B was increased significantly 1 month after TACE (P<0.01). It was concluded that patients with hepatocellular carcinoma had a higher proportion of CD4 CD25 Tregs in peripheral blood. TACE did not significantly affect the level of CD4 CD25 Tregs within short time (such as 1 week). The proportion of CD4 CD25 Tregs in peripheral blood 1 month after TACE was related to the prognosis of hepatocellular carcinoma.     

Targeted induction of differentiation of human bone mesenchymal stem cells into neuron-like cells

A systematic method of isolating and culturing human bone mesenchymal stem cells （hMSCs）, and inducing them to differentiate into neuron-like cells in vitro was established. The hMSCs were isolated from bone marrow with the lymphocyte-separating medium, cultured and expanded in vitro, and induced after addition of compound neuro-revulsants. The morphological changes of hMSCs were observed, and the expression of surface markers in induced hMSCs was immunocytochemically identified during induction period. The hMSCs could be separated, cultured and expanded in vitro. After induction by compound neuro-revulsants for 48 h, the changes of neuron-like cells, such as cellular shrinkage and neurite growth, were observed in some cells. The immunochemical staining revealed nestin （＋） or NF （＋）, and GFAP （-）. It was concluded that hMSCs were successfully cultured and induced to differentiate into neuron-like cells.     

Effect of Noise on the Function of Auditory Brainstem Center

The amplitudes and latencies of auditory brainstem evoked response(ABR)were recorded at both high(50/s)and low(10/s)click rates as sound stimuli before andafter the guinea pigs were exposed to variant intensity noise.The maximum amplitude ofwave V increased significantly,but those of wave Ⅰ,Ⅲ,and Ⅳ decreased significantly afterexposure to 110 dB noise for 20 or 30 min.The wave Ⅴ latency recorded at 50/s clickrate was shorter in every group after exposure.The changes of wave V latency recordedat 10/s click rate were complex,it was shorter after exposure to 95 dB or 105 dB for 10min;no change was found after exposure to 110 dB for 10 min,but it was longer initiallyand then became shorter after exposure to 110 dB noise for 30 min.The difference valueof wave V latency from 10/s to 50/s was negatively correlated with the noise intensityand exposure time.The more intense noise stimuli and the longer time,the less the differ-ence value became.The results indicate that the auditory bralnstem center was affectedby intense noise,and it is helpful to use difference value of wave V latency to analysis thefunction of brainstem center after exposure to noise.     

A new approach to the treatment of malignant effusion.

Lymphocytes isolated from malignant effusion were induced to become LAK cells after in vitro culture with rIL-2. 28 patients with malignant effusion were treated by i.p. or intrapleural administration of autologous LAK cells combined with rIL-2 or by rIL-2 alone. The effusion disappeared in 13 patients and significantly decreased in another 13. Two patients did not respond to the treatment. Tumor cells in effusion disappeared or significantly decreased and lymphocytes significantly increased in all responses. Except for transient fever in 9 patients, no serious side effects were found.

     

The bcl-2 mRNA Expression in GCDC-induced Obstructive Jaundice in Rats and Its Implication in Hepatocellular Apoptosi

The modulatory role of bcl-2 gene in hepatocellular apoptosis of rats with glycochenodeoxycholate (GCDC)-induced obstructive jaundice was investigated. The hepatocytes in normal rats and those with bile duct-ligation for 7 days, 14 days and 21 days were isolated and obtained by in situ collagenase perfusion and primary culture. The expression of bcl-2 mRNA in the hepatocytes was detected by RT-PCR. Primary culture was performed on the hepatocytes from normal rats and those with bile duct-ligation for 14 days. 100 μmol/L GCDC was added to the hepatocytes for incubation for 24 h. The hepatocellular apoptotic ratio was measured by using FCM and hepatocellular apoptosis detected in situ by using TUNEL technique. Results showed that the expression of bcl-2 mRNA was not detectable in the hepatocytes of normal rats by RT-PCR technique, while detectable in the hepatocytes of those with bile duct ligation (BDL) for 7, 14 and 21 days. Hepatocellular apoptosis in the BDL group was obviously decreased as compared with normal control group after addition of 100μmol/L GCDC to the cells for 24 h. It was concluded that the hepatocytes in the BDL rats expressed bcl-2. During obstructive jaundice, expression of bcl-2 from the hepatocytes can inhibit the bile saltinduced hepatocellular apoptosis.     

LIPOPOLYSACCHARIDE-INDUCEDCYTOTOXICITY AGAINST CULTURED MOUSE HEPATOCYTES AND THE ROLE OF NONPARENCHYMAL LIVER CELLS

Lipopolysaccharide(LPS) was found to induce significant hepatocytotxicity against cultured mouse hepatocytes.Degeneration and necrosis of cultured hepatocytes and decerease of hepatocyte viability were prominent.The asparate transferase level and ^3H-TdR release in the medium were significantly increased after treatment,and the degree of these changes paralleled with LPS concentration.Various other parameters showed no significant difference between the hepatocytes cultured alone and those cocultured with nonparenchymal liver cells.However,if the nonparenchymal liver cells were isolated from mice which had been pretreated with D-galactosamine(GalN)not only was the hepatocyotoxicity induced by LPS enhanced,but the cells also showed certain cytotoxicity against cultured hepatocytes even without LPS,These results suggest that nonparenchymal liver cells might promote LPS induced hepatocyte injury.     

Changes of CD4＋ CD25＋ Regulatory T Cells in Peripheral Blood in Patients with Hepatocellular Carcinoma Before and after TACE

This study investigated the changes of CD4＋ CD25＋ regulatory T cells （Tregs） in periph-eral blood of patients with hepatocellular carcinoma before and after transcatheter arterial chemoem-bolization （TACE）. The proportion of CD4＋ CD25＋ Tregs among CD4＋ T lymphocytes in peripheral blood of 33 patients with hepatocellular carcinoma was determined by flow cytometry before, 1 week and 1 month after TACE. And 25 healthy volunteers served as control. One month after TACE, the patients were divided into two groups： 22 in group A, who were in stable condition or getting better; and 10 in group B, who were deteriorating. One patient died and was excluded. The results showed that the percentage of CD4＋CD25＋ Tregs among CD4＋ T lymphocytes did not significantly change in the 33 patients 1 week after TACE as compared with that before TACE, however, the difference was significant （P〈0.01） between the patients with hepatocellular carcinoma and the healthy subjects. The percentage of CD4＋ CD25＋ Tregs among CD4＋ T lymphocytes in group A 1 month after TACE was decreased significantly in comparison with that before and 1 week after TACE （P〈0.01）, whereas, that in group B was increased significantly 1 month after TACE （P〈0.01）. It was concluded that patients with hepatocellular carcinoma had a higher proportion of CD4＋CD25＋ Tregs in peripheral blood. TACE did not significantly affect the level of CD4＋ CD25＋ Tregs within short time （such as 1 week）. The proportion of CD4＋CD25＋ Tregs in peripheral blood 1 month after TACE was related to the prognosis of hepatocellular carcinoma.     

The anti-HSV-2 effect of alumen: In vitro and in vivo experimental studies

This study investigated the anti-HSV-2 effect of alumen through in vitro and in vivo expe-riments.Viable cell counting was employed to assess the toxicity of alumen on Vero cells.The inhibition rate of HSV-2 was defined as the cytopathic effect(CPE)of the cells infected with the virus.Alumen suppositories of different concentrations were vaginally applied to the guinea pigs which were then infected with HSV-2 via a vaginal route.The clinical symptoms were observed and the local virus titer calculated.The results showed that alumen had an in vitro anti-HSV-2 effect by means of antiviral dup-lication,direct killing of the virus,and antiviral adsorption.Alumen suppositories of different concen-trations could reduce or completely inhibit HSV-2 infection in guinea pigs.It was concluded that alumen had an in vitro anti-HSV-2 effect through multiple approaches and it could suppress in vivo vaginal HSV-2 infection of guinea pig to some extent.     

Clinical features and management of Crohn’s disease in Chinese patients

Background An increasing incidence of Crohn‘s disease has been found in China in recent years.Our study has been focused on evaluating the diversity of the clinical manifestations of Crohn‘ s disease in order to improve early diagnostic accuracy and therapeutic efficacy.Methods Thirty patients with active Crohn‘s disease were enrolled and their clinical data, including diagnostic and therapeutic results, were analyzed. Endoscopy combined with histological examination of biopsy specimens provided characteristic features of the disease. Transabdominal bowel sonography (TABS) was used for detecting intestinal complications. Nutritional supportive therapy was given to 20 subjects with active cases of the disease.Results Most patients were young adults with a higher proportion of females to males ( ratio: 1.14:1 ). The disease affects any segment or a combination of segments along with the alimentary tract(from the mouth to the anus). In this study, the colon and small bowel were the major sites involved.Recurrent episodes of abdominal pain in the right lower quadrant and watery diarrhea were the most common symptoms. Granulomas were identifiable in nearly one-third (30. 8%) of all biopsy specimens. In moderate cases of the disease, remission was achieved more quickly through the use of oral prednisone therapy than with SASP or 5-ASA. Beneficial effects on the host‘ s nutritional status were observed. Immunosuppressives were used on an individual basis and showed variable therapeutic effects. Sixteen patients had surgery due to intestinal obstruction or failure to respond to drug therapies. Rapid improvement after surgery was reported.Conclusion Endoscopy (with biopsy) and TABS were both crucial procedures for diagnosis. SASP (or 5-ASA) and prednisone were effective as inductive therapies. Azathioprine has demonstrable benefits after induction therapy with prednisone. Surgery, as an alternative treatment, provided another effective choice in selected patients.     

Mechanism of in Vitro Differentiation of Bone Marrow Stromal Cells into Neuron-like Cells~

In order to study whether marrow stromal cells (MSCs) can be induced into nerve-like cells in vitro, and the mechanism, the MSCs in Wistar rats were isolated and cultured, and then induced with DMSO and BHA in vitro. The expression of specific marking proteins in neurons, glia and neural stem cells were detected before preinduction, at 24 h of preinduction, at 6 h, 24 h, and 48 h of neuronal induction by using immunohistochemistry and Western blotting. The ultrastructural changes after the inducement were observed. The results showed that after the inducement, many MSCs turned into bipolar, multipolar and taper, and then intersected as network structure. At the same time, some MSCs had the typical neuron-like ultrastructure. Immunohistochemistry revealed that NeuN and Nestin expression was detectable after inducement, but there was no GFAP and CNP expression. Western blotting showed the expression of Nestin was strong at 6 h of neuronal induction, and decreased at 24 h, 48 h of the induction. NeuN was detectable at 6 h of neuronal induction, and increased at 24 h, 48 h of the induction. It was concluded MSCs were induced into neural stem cells, and then differentiated into neuron-like cells in vitro.