Objective To investigate the expression of transforminggrowth factor β1(TGF-β1) and insulin-like growth factorⅠ(IGF-Ⅰ)in new bone after low frequency micromovement. Methods Fifteen female sheep from Shandong province were involved in the study and their bilateral tibias transversely osteotomized in the middle shafts with a defect of 2 mm.The hind limbs were fixed with unilateral external fixators connected to a controlled micromovement device. Ten days after osteotomy, one hind limb of each sheep randomlywas selected to perform micromovement at an amplitude of 0.25 mm and a frequency of 1 Hertz, 30 min a day for 4 weeks ( micromovement group). The other hindlimb served as the control group. Five sheep were sacrificed at 3,4 and 6 weeks after osteotomy, respectively, and specimens were harvested for detecting the expression of TGF-β1 and IGF-Ⅰby immunohistochemistry and RT-PCR. Results Immunohistochemistry: In the third postoperative week in the micromovement group, the expression of TGF-β1 was detected in different areas of new chondrocytes at the margin of callus, mainly in proliferating area, and IGF-Ⅰexpressed in osteoblasts at the margin of endochondral ossification area, calcified and mature chondrocytes and osteocytes. There was seldom expression ofIGF-Ⅰ and little expression of TGF-β1 in the corresponding area in the control one. In the 4th postoperative week in the micromovement group, theexpression of TGF-β1 diminished gradually with the mature of new bone and be located in extracellular matrix and osteoblasts around ossified areas; The expression ofIGF-Ⅰ reached the peak and be located mainly in osteoblasts of new bone surface, maturing osteocytes and calcifing osteoid. But there was little expression of them in the control group. In the sixth postoperative week in the micromovement group, there was a little expression of IGF-Ⅰ expression but little expression of TGF-β1; there was nearly no expression of them in the control group. In the micromovement group, the absorbance values of TGF-β1 at 3 and 4 weeksand of IGF-Ⅰat 3, 4 and 6 weeks were significantlyhigher than those in control group（P＜0.05）. RTPCR: In the third and fourth postoperative weeks in the micromovement group, there was higher expression of mRNA of TGF-β1 and TGF-I than those in control group; in the sixth postoperative week, the expression diminished gradually, but was higher than that in control group. The absorbance values of TGF-β1 at 3 and 4 weeks and IGF-Ⅰat 3, 4 and 6weeks were significantly higher than those of control group（P＜0.05）. Conclusion Low frequency and controlled micromovement in the early stage of the fracture healing can promote the expression of TGF-β1 and IGF-Ⅰ.They worked together to regulate the process of the endochondral ossification, while in the late stage the differentiation of osteocytes and mineralization of osteoid were regulated mainly by IGF-Ⅰ, which played an important role in regulating the cell biological behavior during micromovement.
Objective To observe the effect of epidermal growth factor (EGF) on integrin alpha;5 expression and its influence on human retinal pigment epithelium (RPE) cells.Methods Human RPE cells were treated in vitro with 0.1,1.0,10.0,20.0 and 100.0 ng/ml of EGF, the mRNA and protein of integrin alpha;5 was measured by reverse transcription polymerase chain reaction（RT-PCR）and flow cytometry. Human RPE cells were cultured under 4 conditions including DMEM/F12,DMEM/F12+10 ng/ml EGF, DMEM/F12+10 ng/ml EGF+rabbit antihuman integrin alpha;5 antibody (1∶100)，DMEM/F12+10 ng/ml EGF+rabbit antihuman vimentin antibody (1∶100), and their proliferation and migration were measured by methylthiazole tetrazolium（MTT）and Boyden chamber.Results The integrin alpha;5 mRNA level of human RPE cells was not changed after 12 hours of EGF stimulation (F=0.618, P=0.687), however it was induced in a dosedependent manner after 24 and 48 hours of EGF stimulation (F=465.303, 212.340; P=0.000,0.000).The protein level of integrinalpha;5 was higher in 10 ng/ml EGF stimulation compared with the control group and 0.1 ng/ml group（P＜0.01）.MTT and Boyden chamber showed that the integrin alpha;5 expression increased the proliferation and migration of human RPE cells. Conclusion EGF can induce integrin alpha;5 expression,thus increase the proliferation and migration of human RPE cells.
Objective To observe the effect of Twist gene interference on the migration and pAkt protein expression of Rhesus retinal vascular endothelial cell line. Methods The Rhesus retinal vascular endothelial cells (RF/6A) were divided into Twist interference plasmid group, negative control group, and phosphate buffered solution (PBS) group; plasmid vectors were transfected via liposome gene transfection method. Migrated endothelial cells was detected and counted by Transwell chamber assay. Matrigel was used in endothelialcell tube formation; the inhibitory effect of Twist gene interference on endothelial cell tube formation was observed.The effect of Twist gene interference on the expression of pAkt protein in RF/6Acells was measured by Western blot. Results The number of migrated endothelial cells in Twist interference plasmid group was lower than that in the negative control and PBS group (F=23.786，P=0.000).The number of endothelial cell tubes in Twist interference plasmid group was apparently less than that in the negative control and PBS gorup (F=7.159，P=0.014). The expression of pAkt protein in Twist interference plasmid group decreased markedly.Conclusion Twist gene interference can suppress the migration of retinal endothelial cells via inhibiting the expression of pAkt protein.
Objective To verify the effect of palatoplasty with or without velopharyngeal muscular reconstruction on the velar movement through the lateral radiography. Methods From October 1988 to October 2000, 62 patients with cleft palate and velopharyngeal insufficiency were treated. Of them, 32 were repaired by velopharyngeal muscular reconstruction (group A) and 30 by modified von Langenbeck’s procedure (group B). The lateral radiographs and cephalometric analysis were taken. The velopharyngeal closure, velar extensibility, the angle changes of velar elevation, the distance changes of velar levator eminence based on anatomy (LEA) to velopharyngeal closure line (VCL), the varieties in LEA, the comparison of LEA and velar levator eminence based on phonation (LEP), the distance comparison between posterior nasal spine (PNS) to LEA and LEP through soft palate line (SPL) were measured. Results During the phonation, group A was significantly greater than group B in the velopharyngeal closure rate(Plt;0.01)， the velar extensibility(Plt;0.05) and the location comparison between LEA and LEP(Plt;0.01); group B was significantly greater than group A in velar elevation angle (Plt;0.05), the varieties in LEA(Plt;0.05). In velar rest position, the distance of LEA to VCL was greater inB group than in A group(Plt;0.01). There was significant difference in thedistance comparison between LEA and LEP(Plt;0.05), difference between LEA and LEP(Plt;0.01) and the distance PNS-SPL-LEA and PNSSPL-LEP(Plt;0.05) within group B; contrary to the results within group A(P＞0.05). Conclusion The velopharyngeal muscular reconstruction in palatoplasty can result in a near normalizationof anatomic measurement of velar levator muscles and improve the velar functionand velopharyngeal competence. Repositioning of velar muscles in a more anatomic correct, transverse position is more important to improve the velar length andaccordant velar movement with velopharyngeal muscles in functional palatoplasty. The velar angle change and velar elevation during phonation are not determinative factors for velopharyngeal competence.
Sit-stand movement is one of the most common movement behaviors of the human body. The knee joint is the main bearing joint of this movement. Thus, the dynamic analysis of knee joint during this movement has deeply positive influences. According to the principle of moment balance, the dynamics of the knee joint during the movement were analyzed. Furthermore, combined with the data obtained from optical motion capture and six-dimensional ground reaction force test, the curve of knee joint torque was calculated. To verify the accuracy of the analysis of dynamic, the human body model was established, the polynomial equations of angle and angular velocity were fitted according to the experimental data, and the knee joint simulation of the movement was carried out. The result revealed that in terms of range and trend, the theoretical data and simulation data were consistent. The relationship between knee joint torque and ground reaction force was revealed based on the variation law of knee joint torque. During the sit-stand movement, the knee joint torque and the ground reaction force were directly proportional to each other, and the ratio was 5 to 6. In the standing process, the acceleration first increased and then decreased and finally increased in reverse, and the maximum knee torque occurred at an angle of about 140°. In the sitting process, the torque was maximized in the initial stage. The results of the dynamics analysis of knee joint during sit-stand movement are beneficial to the optimal design and force feedback control of seated rehabilitation aids, and can provide theoretical guidance for knee rehabilitation training.
ObjectiveTo observe the effect of bone morphogenetic protein 4 (BMP4) on the proliferation and migration of human retinal microvascular endothelial cells (hRMEC) under oxidative stress. MethodsThe hRMEC cultured in vitro were divided into control group, 4-hydroxynonenal (HNE) treatment group (4-HNE group), 4-HNE+BMP4 group (BMP4 group). Cell culture medium of 4-HNE treatment group was added with 10 μmmol/L 4-HNE; cell culture of BMP4 group was cultured with 10 μmmol/L 4-HNE, and after stimulation for 6 h, 100 ng/ml recombinant human BMP4 was added. The effects of 4-HNE and BMP4 on hRMEC viability was detected by thiazole blue colorimetric method. The effects of 4-HNE and BMP4 on cell migration was determined by cell scratch test. The relative expression of BMP4 mRNA in the cells of the control group and 4-HNE treatment group and the mRNA expression of the control group, the fibronectin (FN) of BMP4 group, laminin (Laminin), α-smooth muscle contractile protein (α-SMA), and collagen type Ⅰ (Collagen Ⅰ), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) were detected by real-time quantitative polymerase chain reaction (qRT-PCR). Western blot was used to detect the relative expression of BMP4 protein in the control group and 4-HNE group. The control group and 4-HNE group were compared by t test. ResultsCompared with the control group, cell viability (t=12.73, 16.26, P=0.000 2, <0.000 1), cell migration rate (t=28.17, 37.48, P<0.000 1, <0.000 1) in 4-HNE group and BMP4 group were significantly increased, and the difference was statistically significant; the relative expression of BMP4 mRNA and protein in the 4-HNE group was significantly increased, and the difference was statistically significant (t=16.36, 69.35, P=0.000 1, <0.000 1). The qRT-PCR test results showed that compared with the control group, the relative expression of VEGF, FN, Laminin, α-SMA, Collagen Ⅰ, and CTGF mRNA in the cells of the BMP4 group was significantly increased, and the difference was statistically significant (t=10.61, 17.00, 14.85, 7.78, 12.02, 10.61, P=0.0004, <0.000 1, 0.000 1, 0.001 5, 0.000 1, 0.000 4). ConclusionBMP4 can induce the proliferation and migration of hRMEC; it can also regulate the expression of angiogenesis factors and fibrosis-related factors in hRMEC.
ObjectiveTo investigate the effect of human subcutaneous adipose-derived stem cells (hADSCs) local transplantation on orthodontically induced root resorption (OIRR) and provide theoretical and experimental basis for the clinical application of hADSCs to inhibit OIRR. MethodsForty 8-week-old male Sprague Dawley rats were randomly divided into experimental group and control group, with 20 rats in each group, to establish the first molar mesial orthodontic tooth movement (OTM) model of rat right maxillary. The rats in the experimental group were injected with 25 μL of cell suspension containing 2.5×105 hADSCs on the 1st, 4th, 8th, and 12th day of modeling, while the rats in the control group were injected with 25 μL of PBS. The rat maxillary models were obtained before and after 7 and 14 days of force application, and 10 rats in each group were killed and sampled after 7 and 14 days of force application. The OTM distance was measured by stereomicroscope, the root morphology of the pressure side was observed by scanning electron microscope and the root resorption area ratio was measured. The root resorption and periodontal tissue remodeling of the pressure side were observed by HE staining and the root resorption index was calculated. The number of cementoclast and osteoclast in the periodontal tissue on the pressure side was counted by tartrate resistant acid phosphatase staining. Results The TOM distance of both groups increased with the extension of the force application time, and there was no significant difference (P<0.05). There was no significant difference in OTM distance between the experimental group and the control group after 7 and 14 days of force application (P>0.05). Scanning electron microscope observation showed that small and shallow scattered resorption lacunae were observed on the root surface of the experimental group and the control group after 7 days of force application, and there was no significant difference in the root resorption area ratio between the two groups (P>0.05); after 14 days of application, the root resorption lacunae deepened and became larger in both groups, and the root resorption area ratio in the experimental group was significantly lower than that in the control group (P<0.05). The range and depth of root absorption in the experimental group were smaller and shallower than those in the control group, and the root absorption index in the experimental group was significantly lower than that in the control group after 14 days of force application (P<0.05). The number of cementoclast in the experimental group was significantly lower than that in the control group after 7 and 14 days of force application (P<0.05); the number of osteoclasts in the experimental group was significantly lower than that in the control group after 14 days of force application (P<0.05). Conclusion Local transplantation of hADSCs may reduce the area and depth of root resorption by reducing the number of cementoclasts and osteoclasts during OTM in rats, thereby inhibiting orthodontic-derived root resorption.
ObjectiveTo address the effect and mechanism of interleukin 17 (IL-17) on the proliferation, migration and apoptosis of human retinal vascular endothelial cells (HREC). MethodsIL-17 receptor (IL-17R) mRNA and protein expression in human retinal vascular endothelial cells (HREC) were quantified by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation of HREC was examined using CCK-8 assay in the presence of different concentrations of IL-17. Cell migration of HREC was detected using wound scratch assay. Flow cytometry was used to test the effect of IL-17 on the apoptosis of HREC. The effects of IL-17 on HREC expression of basic fibroblast growth factor (bFGF), Caspase-3 and thrombospondin-1 (TSP-1) were quantified by reverse transcriptase polymerase chain reaction (RT-PCR). The effect of IL-17 on HREC expression of Caspase-3 was examined using Western blot. ResultsIL-17 receptor (IL-17R) expressed in HREC as quantified by RT-PCR and Western blot. The proliferation of HREC in the presence of IL-17 was promoted in a dosage-dependent manner (t=-3.235, -6.276;P=0.032, 0.000). Wound scratch assay showed a significant increase in the migrated distance of HREC with IL-17 stimulation under the concentration of 100μg/L(t=-3.551, -2.849; P=0.006, 0.019), 200μg/L(t=-10.347, -4.519; P=0.000, 0.001) and 500μg/L (t=-3.541, -2.607; P=0.008, 0.036). The intervention of 200μg/L IL-17 can effectively inhibit the apoptosis of HREC, compared with the control group using flow cytometry (t=5.682, P=0.047). RT-PCR results showed that IL-17 can promote the expression of bFGF and inhibit the expression of Caspase-3 and TSP-1. Western blot result also showed that IL-17 can suppress the protein expression of Caspase-3. ConclusionThe mechanism of IL-17 promoting proliferation, migration but suppress apoptosis of HREC may via regulating the expression of bFGF and Caspase-3.
ObjectiveTo investigate the expression of miRNA-1 in denervated skeletal muscle at different periods, and to explore effects of passive movement on the expression of miRNA-1 and differentiation of myoblasts in denervation-induced skeletal muscle atrophy in rats. MethodsTwenty-seven Sprague Dawley rats, weighing (200±10) g, were randomly divided into sham-operated group (group A, n=3), denervated group (group B, n=12), and passive movement group (group C, n=12). After the right sciatic nerve was exposed and dissociated, the sciatic nerve of 1 cm in length was removed in groups B and C; resection was not performed in group A. At 1 day after operation, passive flexion and extension movement was performed on the right hind limb in group C. At 6 hours in group A and at 3, 7, 14, and 28 days in groups B and C, 3 rats were sacrificed to measure the wet weight ratio of gastrocnemius muscle, to observe the diameter of the gastrocnemius muscle cell and evaluate the muscle atrophy by HE staining; RT-PCR was used to detect the mRNA expression of miRNA-1 and myocyte differentiation factor (MyoD), and immunohistochemistry to determine the protein expression of MyoD. ResultsAtrophy in various degrees was observed in denervated gastrocnemius muscle of groups B and C. The muscle fiber arranged in disorder and the diameter of the muscle cells decreased gradually with the time, without normal structure and morphology. The wet weight ratio and the cell diameter of the gastrocnemius in groups B and C were significantly less than those in group A (P<0.05); the wet weight ratio at 7, 14, 28 days and the cell diameter at 7, 14 days of group B were significantly greater than those of group A (P<0.05). The expressions of miRNA-1 and MyoD mRNA gradually increased with time in groups B and C, but were significantly less than those of group A at each time point (P<0.05). At 7, 14, and 28 days after operation, the expressions of miRNA-1 and MyoD mRNA in group C were significantly higher than those in group B (P<0.05). Immunohistochemical staining showed positive expression of MyoD in groups A, B, and C at each time point, but higher expression was observed in groups B and C than group A; the expression increased with time in groups B and C, and it was significantly higher in group C than group B. The correlation analysis results showed that the overall change trend of miRNA-1 and MyoD had no relation with the gastrocnemius wet weight ratio at 3 and 7 days (P>0.05), and had positive correlation at 14 and 28 days (P<0.05); positive correlation was found between the relative expression of MyoD and miRNA-1 mRNA (P<0.05). ConclusionPassive movement can prevent amyotrophy by increasing the expression of miRNA-1 and promoting the differentiation of myoblasts.
The extraction of pulse rate variability(PRV) in daily life is often affected by exercise and blood perfusion. Therefore, this paper proposes a method of detecting pulse signal and extracting PRV in post-ear, which could improve the accuracy and stability of PRV in daily life. First, the post-ear pulse signal detection system suitable for daily use was developed, which can transmit data to an Android phone by Bluetooth for daily PRV extraction. Then, according to the state of daily life, nine experiments were designed under the situation of static, motion, chewing, and talking states, respectively. Based on the results of these experiments, synchronous data acquisition of the single-lead electrocardiogram (ECG) signal and the pulse signal collected by the commercial pulse sensor on the finger were compared with the post-auricular pulse signal. According to the results of signal wave, amplitude and frequency-amplitude characteristic, the post-ear pulse signal was significantly steady and had more information than finger pulse signal in the traditional way. The PRV extracted from post-ear pulse signal has high accuracy, and the accuracy of the nine experiments is higher than 98.000%. The method of PRV extraction from post-ear has the characteristics of high accuracy, good stability and easy use in daily life, which can provide new ideas and ways for accurate extraction of PRV under unsupervised conditions.