To study and optimize the fermentation parameters for expressing human-like collagenⅡduring E. coli high-density fermentation. The effects of pH, temperature, dissolved oxygen and induction instant on the cell growth and human-like collagenⅡproduction were investigated to optimize the fermentation conditions. The results demonstrated that the following conditions were beneficial for cell growth and foreign gene expression, controlling pH in phase induction at 6.8 and initial pH at 6.5, maintaining fermentation temperature and dissolved oxygen concentration was controlled at 34?C and 20% respectively, and implementing induction at the later logarithmic growth phase. Under the optimized condition, the cell density and human-like collagenⅡyield could reach 88.4 g/L and 14.2 g/L, respectively.

Keratoconus is a chronic non-inflammatory corneal disease characterized by thinning and localized protrusion of the corneal stroma in a conical shape. It often leads to irreversible irregular astigmatism and varying degrees of decline in corrected visual acuity. With the rise of femtosecond laser refractive surgery, corneal lenses were discovered as a good biological material. Corneal lens implantation of the cornea can increase the thickness of the cornea and delay the progression of keratoconus disease for further treatment. Currently, convex lenses obtained from myopic small incision lenticule extraction(SMILE)surgery and concave lenses obtained from farsighted SMILE surgery, as well as parallel lenses obtained from donors, can be implanted into the cornea with the aid of femtosecond lasers as lens materials. This article will summarize and discuss the above three lens material implantation methods to provide reference for the selection of keratoconus treatment.

Objective: To investigate the effects of human adipose-derived mesenchymal stem cells (ADSCs) and platelet-rich plasma (PRP) on healing of wounds with full-thickness skin defects in mice. Methods: ADSCs were isolated from the lumbar and abdominal fat donated voluntarily by a healthy woman undergoing liposuction in the Department of Plastic Surgery of Guangzhou General Hospital of Guangzhou Military Area Command, and the cells were cultured and identified. ADSCs of the second passage were used in the following experiments. The venous blood of the volunteer was taken, and PRP was obtained by secondary centrifugation. Thirty-six C57BL/6 mice were divided into simple injury group (n=12), simple ADSCs treatment group (n=12), and ADSCs+ PRP treatment group (n=12) according to the random number table. Each mouse was inflicted with a 1 cm×1 cm wound with full-thickness skin defect on the back. Immediately after injury, the wounds of mice in simple injury group were subcutaneously injected with 1 mL normal saline, the wounds of mice in simple ADSCs treatment group were subcutaneously injected with 1 mL phosphate buffer solution-blended ADSCs suspension (with concentration of 5×10⁵ /mL, the same below), and the wounds of mice in ADSCs+ PRP treatment group were subcutaneously injected with 1 mL mixture of PRP and ADSCs (1∶2 volume ratio). Three mice in each group were taken on post injury day (PID) 3, 5, 7, and 14 to observe the gross condition of wound, and the wound healing rate was calculated. On PID 3, 5, and 7, the non-healing wound tissue and 0.5 cm normal skin tissue around the wound margin were taken after gross observation. The inflammation, re-epithelialization, and angiogenesis of tissue were observed by hematoxylin and eosin staining, and the re-epithelialization rate was calculated. The collagen synthesis of tissue was observed by masson staining. Immunohistochemistry was used to observe the expression of macrophages of tissue samples collected on PID 3 and 5. Data were processed with analysis of variance of factorial design and Least-Significant Difference test. Results: (1) On PID 3, the wounds of mice in ADSCs+ PRP treatment group were with granulation tissue regeneration, redness, and swelling, and the wounds of mice in the other two groups were ruddy and with effusion. On PID 5, the wounds of mice in ADSCs+ PRP treatment group had less redness and swelling, which were dry with obvious scab, and wounds of mice in the other two groups were obviously red and swollen. On PID 7, scab formed basically on wounds of mice in the three groups. On PID 14, the wounds of mice in the three groups basically healed, and their crusts were off. On PID 3, 5, 7, and 14, the wound healing rates of mice in ADSCs+ PRP treatment group were obviously higher than those of the other two groups (P<0.05 or P<0.01). On PID 5 and 7, the wound healing rates of mice in simple ADSCs treatment group were obviously higher than those of simple injury group (P<0.01). (2) On PID 3, granulation tissue regeneration of wounds in ADSCs+ PRP treatment group was more than that in the other two groups. On PID 5, inflammatory reaction of wounds of mice was mild in ADSCs+ PRP treatment group, which was severe in the other two groups. On PID 7, the re-epithelialization process of wounds of mice was almost completed in ADSCs+ PRP treatment group, and the number of new vessels was more in ADSCs+ PRP treatment group than in the other two groups. The migration distance of regenerated epithelia around the wound edge in simple injury group and simple ADSCs treatment group was short. On PID 3, 5, and 7, the re-epithelialization rates of wounds of mice in ADSCs+ PRP treatment group were (37.6±4.5)%, (59.1±1.3)%, and (89.2±4.3)%, respectively, significantly higher than (25.7±1.5)%, (34.5±4.4)%, and (50.8±2.7)% in simple injury group and (29.1±0.8)%, (42.6±2.9)%, and (72.9±3.0)% in simple ADSCs treatment group (P<0.01). On PID 5 and 7, the re-epithelialization rates of wounds of mice in simple ADSCs treatment group were significantly higher than those in simple injury group (P<0.05 or P<0.01). (3) On PID 3 and 5, a quite large number of new collagen fibers appeared in granulation tissue of wounds of ADSCs+ PRP treatment group, while the collagen fibers in the other two groups were less. On PID 7, the granulation tissue of mice in ADSCs+ PRP treatment group decreased, and a large number of new collagen fibers appeared. The collagen fibers in wounds tissue of mice in simple ADSCs treatment group increased, while the collagen fibers deposited in wounds tissue of mice in simple injury group was still less. (4) On PID 3 and 5, the numbers of macrophages in wounds tissue of mice in simple ADSCs treatment group were 4.7±0.6 and 5.3±0.6 respectively, obviously lower than 6.3±0.6 and 7.7±0.6 in injury group (P<0.05 or P<0.01); the numbers of macrophages in wounds tissue of mice in ADSCs+ PRP treatment group were 3.0±1.1 and 2.7±0.5, significantly lower than those in the other two groups (P<0.05 or P<0.01). Conclusions Human PRP and ADSCs are involved in the early inflammation, metaphase of tissue proliferation, and re-epithelialization and shaping process of late stage of wounds with full-thickness skin defects in mice. The combination of ADSCs and PRP may be a comparatively good combination to improve the speed and quality of wound healing.

Objective To investigate the changes of mestruation patterns and adverse effects during the treatment of levonorgestrel-releasing intrauterine system (LNG-IUS) for symptomatic adenomyosis in a prospective cohort study. Methods From December, 2006 to December, 2014, patients of symptomatic adenomyosis diagnosed by transvaginal ultrasound in Peking Union Medical College Hospital were given LNG-IUS. Before and after placement of IUS, all patients′ parameters were recorded, including carrying status of IUS, symptoms and scores of dysmenorrhea, menstruation scores, biochemical indicators, physical parameters, menstruation patterns and adverse effects. Risk factors for changes of menstruation patterns and adverse effects, and their impact on treatment effects were analyzed. Results Totally 1 100 cases met inclusion criteria, with median age 36 years (range 20-44 years), median follow-up 35 months (range 1-108 months). During follow-up changes of menstruation patterns increased significantly with amenorrhea and shortened-menstruation being the most common manifestations. On 3, 6, 12, 24, 36, 48 and 60 months after the placement of LNG-IUS, 0, 5.8%(43/744), 6.9%(47/682), 10.1%(60/595), 17.3%(87/502), 27.2%(104/383) and 29.6%(82/277) patients achieved amenorrhea respectively (P<0.01). Total and subclassification of adverse effects decreased significantly (P<0.01). Within 12 months and >12 months after placement, abdominal pain and body weight increasing ≥5 kg/year were the most common adverse effects. Changes of menstruation patterns, total and subclassifications of adverse effects were neither dependent on patient parameters, treatment modes and treatment effects, nor could predict future LNG-IUS carrying status (all P>0.05). After taking out of LNG-IUS, most changes of menstruation and adverse effects disappeared. Conclusions During the treatment of LNG-IUS for symptomatic adenomyosis, changes of menstruation patterns increase gradually with amenorrhea and shortened-menstruation being the most common manifestations, while adverse effects decrease significantly. Changes of menstruation patterns or adverse effects neither have any risk factor nor have impact on treatment effects.

Objective: To understand literacy development of school-aged children of grade 2-5 and its influencing factors in Guangzhou,and to provide a reference for the reform of the teaching of Chinese subject. Methods: By using cluster sampling method, 1 661 school-age children from grade 2 to grade 5 from 5 primary schools in Guangzhou were selected. The self-designed questionnaire was used to obtain demographic data of the participants. The Primary School Literacy Assessment Scale and the Pupil Rating Scale Revised Screening for Learning Disabilities was used to evaluate the literacy and the cognitive characteristics of participants. Results: The average literacy of children of grade 2 to grade 5 in Guangzhou was as follows: grade 2 was (1 159±295), grade 3 was (1 919±394), grade 4 was (2 599±365), grade 5 was (2 947±303), higher than the norm(P<0.01). The average literacy of grade 2 students was lower than the national curriculum requirements while students of grade 3 to grade 5 met the requirements. Univariate analysis found differences in literacy among school-age children based on gender, reading experience before age 6, and parental education background (P<0.01). Multiple linear regression analysis revealed that grade (β=607.04), preschool reading experience (β=109.89), father’s education (β=27.14), language factor (β=27.21), social behavior (β=16.03) was positively correlated with literacy in boys (P<0.05). Grade (β=603.53), auditory comprehension and memory (β=29.39), language factor (β=16.74) was positively correlated with the literacy of girls (P<0.05), while time and orientation judgments were negatively correlated with the literacy of both boys (β=-18.95) and girls (β=-21.93) (P<0.05). Conclusion The literacy level of school-age children in grade 2 to 5 in Guangzhou has reached the national literacy requirements with students in grade 2 being relatively lower. Literacy is related to grade, preschool reading experience, father’s education, and child’s cognitive characteristics. Factors affecting literacy in boys and girls are different. Literacy education should vary according to gender.

Objective: This study aims to explore the influence of birth order and age difference on the emotional and behavioral problems among children from two-child family, so as to provide advice on parenting. Methods: A total of 482 students in grades 2 to 5 from the 5 primary schools in Guangzhou were selected, all of whom came from two-child families (excluding twin children). Emotional and behavioral problems were investigated by using the Conner’s Parental Symptom Questionnaire (PSQ). Results: Among children from two-child families, higher prevalence of behavioral problems, learning problems, impulsive hyperactivity and hyperactivity index in older compared with younger children (β=-0.09，-0.16，-0.09，-0.10,P<0.05). The scores of behavioral problems and hyperactivity index were significantly higher among those of children who were 6-9 years older than young siblings, compared to those who were 0-3 years older than younger siblings(β=0.20,0.21,P<0.05). No similar pattern was found among the younger siblings(P>0.05). Conclusion Among school-age children from two-child families, emotional and behavioral problems of the elder ones were more serious than the younger ones’. Age difference between siblings shows impact on the emotional and behavioral development among elder children, however no significant impact is noticed among the younger ones.

Objective To investigate treatment effects of levonorgestrel-releasing intrauterine system (LNG-IUS) for adenomyosis with severe dysmenorrhea in a prospective cohort study. Methods From December 2006 to December 2014, patients of symptomatic adenomyosis diagnosed by transvaginal ultrasound in outpatient or inpatient clinics of Peking Union Medical College Hospital were given the treatment of LNG-IUS. Before and after placement of LNG-IUS, all the patients′parameters were recorded prospectively, including symptoms and scores of dysmenorrhea, menstruation scores, biochemical indicators, physical parameters, carrying status of LNG-IUS, menstruation patterns and adverse effects. Changes of scores and patterns of pain during follow-up were analyzed. Results Totally 1 100 women meets inclusion criteria, among which 640 cases (58.18%, 640/1 100) had severe dysmeorrhea, with median follow-up period of 35 months (range 1-60 months), and accumulative carrying rate of 65% at 60 months follow-up. After placement of LNG-IUS, scores of pain and ratio of severe dysmenorrhea had decreased significantly compared with baselines (all P<0.01), the scroes of visual analog scale (VAS) were 8.1 ± 0.9, 5.5 ± 2.4, 4.6 ± 2.4, 3.3 ± 2.2, 2.2 ± 2.1, 2.2 ± 1.8, 1.4 ± 1.6 and 1.3 ± 1.3 at 0, 3, 6, 12, 24, 36, 48 and 60 months respectively. During 36 months after placement of LNG-IUS, scores of pain had improved significantly compared with preceding period (all P<0.01). We found no universal dependent factors predicting improvement of pain, which was neither relevant with simultaneous changes of menstruation patterns nor adverse effects (all P>0.05). Conclusion LNG-IUS is effective for adenomyosis of severe dysmenorrhea. Improvement of pain is independent on patients characters, menstruation patterns or adverse effects.

Objective To investigate treatment effects of levonorgestrel-releasing intrauterine system (LNG-IUS) for adenomyosis with menorrhea in a prospective study. Methods From December 2006 to December 2014, patients of symptomatic adenomyosis diagnosed by transvaginal ultrasound in outpatient or inpatient clinics of Peking Union Medical College Hospital were given the treatment of LNG-IUS. Before and after placement of LNG-IUS, all the patients′parameters were recorded prospectively, including scores of menstruation blood loss, carrying status of IUS, symptoms and scores of dysmenorrhea, biochemical indicators, physical parameters, menstruation patterns and adverse effects. Changes of pictorial chart scores of menstruation and distribution of anemia during follow-up were analyzed. Results Totally 1 100 women meets inclusion criteria, among which 618 cases (56.18%, 618/1 100) had severe menorrhea, with median follow-up period of 28 months (range 1-60 months), and accumulative carrying rate of 66% at 60 months follow-up. After placement of LNG-IUS, compared with baselines, pictorial chart scores and ratio of menorrhea had decreased significantly (all P<0.01), the scroes of menstruation were 157±34, 94±35, 70±33,67 ± 18, 67 ± 20, 65 ± 19, 66 ± 19, 65 ± 21 at 0, 3, 6, 12, 24, 36, 48 and 60 months respectively. During 24 months after placement of LNG-IUS, pictorial chart scores and distribution of anemia had improved significantly compared with preceding period (all P<0.01). We found no dependent factors predicting improvement of pictorial chart scores of menorrhea, which was neither relevant with simultaneous changes of menstruation patterns nor adverse effects (all P>0.05). Conclusions LNG-IUS is effective for adenomyosis of menorrhea. Improvement of menstruation blood loss is independent on patients characters, menstruation patterns or adverse effects.

In comparison with the traditional two-dimensional tumor cell culture, the three-dimensional tumor spheroid culture can not only provide with an in vivo-like growth environment for tumor cells, but also maintain maximum cell activities. Therefore, the three-dimensional tumor spheroid culture is widely used in oncology research. In particular, the three-dimensional tumor cells retain the material and structural basis of the microenvironment of tumor in vivo, which is closer to the actual physiological environment, allowing it to be an ideal in vitro model for evaluating the tumor treatment response and drug resistance in tumors. This review summarizes the mechanisms of drug resistance in three-dimensional tumor cells, especially those induced by the morphology and microenvironment of three-dimensional tumor spheres, and puts forward the problems existing in the current three-dimensional tumor cells model, as well as the future development direction.