CD72 is a B cell specific receptor that exists in multiple alternative splicing forms. Eight novel alternative splicing forms of CD72 were identified from the spleenocytes of BALB/C mice. Two very unique intron sequences were found in those alternative splicing forms. One kind of splicing variants retained the intron1 in the mRNA. This intron can be translated into 32 amino acid residues without changing the reading frame of the whole proteins. Another kind of splicing variants used an alternative 3' splice site in intron 3(3'AS) which led to premature termination of its encoded protein. The differential expression of the CD72 splicing variants were compared in BALB/C and NZB/W mice that were at different stage of systematic lupus erythematosis(SLE) disease development. It was found that 1) splicing forms containing 3'AS was rare in all samples examinated; 2) splicing forms containing two ITIM domains and transmembrane domains were more abundant in BALB/C mice than in NZB/W mice, even in some cases the two ITIM domains were separated by the intron 1; 3) a shorter splicing form with both exon2 and exon3 missing was expressed highly in terminally diseased NZB/W mice.These results suggested an important role of CD72 alternative splicing forms in B cell receptor signaling and in SLE.

The vectors play an important role in gene therapy by transporting the therapy genes into target tumor cells. At present there are mainly two kinds of gene vectors, including biological and non-biological vectors. Biological vectors, including virus, bacteria and stem cells, have good target activity and can efficiently delivery the genes into target tumor cells, but have potential genetic hazards.However, non-biological vectors, including liposomes and nanoparticles, can be simplely prepared with high genetic safety, but they can not transport the genes efficiently. Therefore, establishing safe and effective malignant glioma gene treatment system and finding gene vectors with good target activity become the research focus.

Objective To establish D-dimer normal reference range in pregnant and postpartum women in Zhejiang Han population.Methods Plasma samples were collected from 669 healthy pregnant women, 578 healthy postpartum women, 8 venous thrombosis or DIC patients and 80 healthy non-pregnant women in Women′s Hospital, School of Medicine, Zhejiang University from March 2009 to August 2010.According to different gestational week, postpartum days and delivery pattern, the healthy pregnant and postpartum women were stratified into 3 groups: (1) ≤13 weeks (n=120), 14-20 weeks (n=120), 21-27 weeks (n=145), 28-34 weeks (n=147), ≥35 weeks (n=137);(2) The first day after vaginal delivery (n=163), the second day after vaginal delivery (n=121);(3) The first day after cesarean sections (n=166), the second day after cesarean sections (n=128). These groups were further stratified based on age: ＜30 years old and ≥30 years old.All blood samples were drawn in citrate sodium anticoagulated blood.D-dimer concentration was determined by STA-R Evolution coagulation analyzer.Since D-dimer concentration showed non-normal distribution, the normal values (one-tailed) were established by using (P95) percentile method.The results of 8 patients were used to validate the established normal values.Results In the group of ＜30 years old, the D-dimer values[M(P25-P75)]in group of ≤13 weeks, 14-20 weeks, 21-27 weeks, 28-34 weeks, ≥35 weeks and healthy non-pregnant women were 0.25(0.17-0.37), 0.51(0.38-0.75), 0.75(0.57-1.10), 1.14(0.80-1.48), 1.60(1.14-1.89) and 0.20(0.10-0.28) mg/L, respectively, which showed statistical difference(H=239.24, P＜0.05).In the group of ≥30 years old, the D-dimer values of the above different groups were 0.28(0.14-0.38), 0.50(0.36-0.65), 0.83(0.59-1.41), 0.93(0.68-1.37), 1.47(1.22-1.84) and 0.17(0.12-0.25) mg/L, respectively, which also showed statistical difference(H=127.75, P＜0.05).In the group of ＜30 years old, the D-dimer values were 2.45(1.51-3.77), 1.30(0.97-1.96), 2.68(1.52-3.74) and 1.55(1.10-2.10) on the first and second day after vaginal delivery and cesarean section, respectively, which showed statistical difference (H=64.85,P＜0.05).In the group of ≥30 years old, the corresponding values were 2.20(1.33-3.54), 1.33(1.02-2.14), 2.27(1.66-3.17) and 1.62(1.26-2.69), respectively, which also showed statistical difference(H=18.64, P＜0.05).D-dimer normal values were established based on different gestational week, postpartum days and delivery pattern.The normal values of ≤13 weeks, 14-20 weeks, 21-27 weeks, 28-34 weeks and ≥35 weeks were ≤0.64 mg/L, ≤1.54 mg/L, ≤2.60 mg/L, ≤3.01 mg/L and ≤3.19 mg/L, respectively. The normal values of 1st day after vaginal delivery, 2nd day after vaginal delivery,1st day after cesarean sections and 2nd day after cesarean sections were ≤7.83 mg/L, ≤3.29 mg/L, ≤9.95 mg/L and ≤3.80 mg/L.All 8 patients showed positive results with the above normal values.Conclusion D-dimer normal values in pregnant and postpartum women in Zhejiang Han population are established, which can improve the application values of D-dimer in the pregnant and postpartum population.

OBJECTIVE:To observe analgesic effect and safety of ketorolac tromethamine combined with butorphanol tartrate in the treatment of acute pain after fracture surgery. METHODS:76 acute pain patients after fracture surgery were selected and ran-domly divided into control group and observation group,with 38 cases in each group. Control group was given Ketorolac trometh-amine injection 30 mg,ivgtt,and then 2 ml/h,0.5 mg/kg,ivgtt;observation group was additionally given butorphanol tartrate 10 mg,ivgtt,on the basis of control group. Pain degree was evaluated with VAS before and 10 min,1,2,4 and 6 h after treatment, and the occurrence of ADR was observed in 2 groups. RESULTS:10 min,1,2,4 and 6 h after treatment,VAS score of 2 groups were significantly lower than before,with statistical significance(P<0.05);6 h after treatment,VAS score of observation group was significantly higher than that of control group,with statistical significance (P<0.05);there was no statistical significance in VAS score between 2 groups 10 min,1,2 and 4 h after treatment (P>0.05). The incidence of ADR in observation group (5.26%)was significantly lower than in control group(21.05%),with statistical significance(P<0.05). CONCLUSIONS:Com-pared with ketorolac tromethamine alone,ketorolac tromethamine combined with butorphanol tartrate shows shorter analgesia dura-tion,similar therapeutic efficacy,and lower incidence of ADR.

Objective To knockout Rag2 and IL2rg genes and construct severe combined immunodeficiency mice based on CRISPR/Cas9 technology. Method Design and synthesis of 25 bp sgRNA were made according to the Rag2 and IL2rg sequences in Genbank. After annealing, sgRNA was cloned into pX330 vector. Recombination plasmid Rag2?sgRNA, IL2rg?sgRN and Cas9 were then transcribed into RNA, these RNA were microinjected into zygotes and the zygotes were transplanted into recipient ICR mice. F0 founders were born and mutated F0 founders mated with wild type mice to obtain F1 generation heterozygous mice. Mutated F1 mice were crossed and got F2 generation homozygous mice. Genotype and phenotype of the knockout mice were identified by sequencing, flow cytometry and xenograft model. Results Rag2?sgRNA and IL2rg?sgRNA recombination plasmids were constructed and transcribed into RNA. After microinjection and mat? ing, F0 founders were born and F2 homozygous mice were obtained. The results of sequencing showed that there were two types of genotype in IL2rg gene, 10 bp or 11 bp deletion;however, there was only one genotype in Rag2 gene, which was 8 bp deletion. Compared with wild?type BALB/c mice, the number of CD3 +, B220 + and NKp46 + cells in peripheral blood of the knockout mice was reduced significantly. After inoculation of human breast cancer cell line SKBR?2HL cells, tumor size in the xenograft mouse model was increased gradually along with time extension. Conclusion CRISPR/Cas9 is an efficient way to mutate Rag2 and IL2rg gene in mice in vivo, leading to aberrant T cells, B cells and NK cells.

Objective To establish a cultivating method for obtaining a large number of P0 generation human placental chorionic-derived mesenchymal stem cells (hpcMSCs).Methods The hpcMSCs were isolated from human placental chorion.After primary culturing and culturing for seven days,the culture medium,the non-adherent tissue and the douching normal saline of the primary culture were centrifuged and re-cultured twice.Cell morphology was observed by an inverted microscope.CCK-8 was used to measure the cell growth curve.Flow cytometry was used to detect cell surface markers.Adipogenic and osteogenic differentiation kits were used to assess the cell differentiation potential.Results The obtained hpcMSCs were fibroblast-like adherent cells and (25.54±3.38)×106 cells were obtained per placenta.The total yield of the primary culture,secondary culture and tertiary culture were (11.73±2.09)×106,(11.12±1.42)×106 and (2.69±0.71)×106,respectively,and the incubation time were (12.00±0.64) d,(8.87±0.63) d and (12.33±0.80) d.There was significant differences in incubation time between the secondary culture and the primary culture as well as the tertiary culture (all P＜0.05),and there was no significant difference between the primary culture and the tertiary culture.However,the incubation time of the tertiary culture had an increasing trend (P＞0.05).The yield per culture flask of the primary culture,secondary culture and tertiary culture were (1.12±0.15) × 106,(2.10±0.16)×106 and (1.04±0.16)×106,respectively.There was significant differences in the yield per culture flask between the secondary culture and the primary culture as well as the tertiary culture (all P＜0.05),and there was no significant difference between the primary culture and the tertiary culture.However,the yield per culture flask of the tertiary culture had a decreasing trend (P＞0.05).There was no difference among the three cultures in the growth curve and the expression of surface markers,and the osteogenic and adipogenic differentiation were all positive.Conclusions The P0 generation hpcMSCs isolated from a choriocarcinoma sample can be doubled by the three cultures compared with the primary culture,which can provide plenty stem cell source for the regenerative medicine.

BACKGROUND:There are a lot of studies on isolation and culture methods of human placental chorionic-derived mesenchymal stem cells (hpcMSCs), but how to simply and efficiently harvest a large amount of primary MSCs has not been resolved. OBJECTIVE:To optimize the tissue explants method of isolating and culturing hpcMSCsin vitro. METHODS:Human placental chorionic villi were collected from full-term deliveries under aseptic condition and isolated by electric homogenizer. hpcMSCs were prepared by tissue explants method. The fluid and tissue of the primary culture flask and douching normal saline of the initial culture were centrifuged and prepared for secondary culture. RESULTS AND CONCLUSION: It saved time and effort to treat human placental chorionic villi with electric homogenizer, with good effects on tissue dispersion and removal of red blood cells. The average time of cell acquisition in initial culture and secondary culture was (17.73±1.14) and (10.03±1.30) days, respectively. The yields of primary cultured cells in initial culture and secondary culture were (6.97±0.98)×105 and (13.82±1.44)×105per Φ100 mm culture dish, respectively. The adherent cells showed fibroblast-cell-like shape, which were in parallel or circinate arrangement. Highly expressed CD73, CD105 and CD90 could be detected in the third generation of hpcMSCs, but CD34, CD45, CD14, CD19 and HLA-DR were negative. Following induction, alizarin red staining and oil red O staining produced a strong reaction in cells. In a word, the optimized method is a simple and efficient method for obtaining a large amount of primary hpcMSCs.

OBJECTIVETo assess the accuracy of ultrasound-guided 16G and 18G core needle biopsy for detecting ultrasound visible breast lesions with different sonographic features.

METHODSA total of 955 sonographically detected breast lesions examined with ultrasound-guided core needle biopsy (US-CNB) and subsequently surgically excised from July 2005 to July 2012 were retrospectively reviewed. Histological findings of US-CNB and the surgical specimens were analyzed for agreements, sensitivities, false negative rates, and underestimate rates according to different sonographic features.

RESULTSThe pathological results of the US-CNB showed malignant lesions in 84.1%, high-risk lesions in 8.4%, and benign lesions in 7.5% of the samples. The overall agreement rates were 92.4% for 16G CNB and 92.8% for 18G CNB; their complete sensitivities and false negative rates were both 98.6% and 1.4%, respectively; the high-risk underestimate rates and DCIS underestimate rates were 48.0% and 46.2% for 16G CNB vs 53.3% and 41.2% for 18G CNB, showing no significant difference between the two groups (P>0.01). For both 16G and 18G CNB, the agreements were better for mass lesions than for non-mass lesions (P<0.01). For the mass lesions with a diameter no greater than 10 mm, the agreement rates were lower than the overall data (P<0.01). Calcification in the lesions did not affect the agreement rates (P>0.01).

CONCLUSIONUltrasound-guided 16G and 18G CNB are both accurate methods for evaluating ultrasound visible breast mass lesions with a diameter larger than 10 mm.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Biopsy, Fine-Needle ; instrumentation ; methods ; Biopsy, Needle ; instrumentation ; methods ; Breast ; pathology ; Female ; Humans ; Middle Aged ; Sensitivity and Specificity ; Ultrasonography, Mammary ; Young Adult

Objective To investigate the short-term clinical efficacy of arthroscopic reconstruction of posterior cruciate ligament (PCL) via double posteromedial portals.Methods A retrospective case series study was performed on the clinical data of 29 patients with PCL injury from January 2013 to January 2018 admitted to Xuancheng People's Hospital of Anhui province.There were 15 males and 14 females,with an average age of 49.6 years (range,27-61 years).There were 13 patients with left knee injury and 16 patients with right knee injury.The combined injuries included five patients with medial meniscus tear,seven patients with lateral meniscus tear,one patient with medial collateral ligament injury,and two patients with lateral medial collateral ligament injury.The average duration from injury to surgery was 14.1 days (range,5-97 days).All patients received arthroscopic PCL reconstruction surgery using double posteromedial portals.The operation time and intraoperative blood loss were recorded.The posterior drawer test,reverse Lachman test,posterior translation,and Lysholm knee score scale were used to evaluate knee function preoperatively and 6 months after operation.The complications were recorded.Results All patients were followed up for 6-22 months (mean,12 months).The operation time was (63.3 ±9.5) minutes,and the intraoperative blood loss was (48.9 ± 22.8)ml.The posterior drawer tests and reverse Lachman tests of all patients turned to be negative 6 months after surgery.Preoperatively,the posterior translation and Lysholm score were (11.2 ± 3.3)mm and (42.7 ± 12.2)points,respectively.At 6 months postoperatively,the posterior translation and Lysholm score were (2.2 ± 0.5) mm and (86.3 ± 9.0) points,respectively (P ＜ 0.05).No complication such as infection and neurovascular injury occured.Conclusion The technique of arthroscopic PCL reconstruction surgery using double posteromedial portals has the advantages of good knee joint stability,less tibial posterior translation,and fast function recovery with satisfactory short-term efficacy.

Objective:To observe the effect of autologous fat filling combined with botulinum toxin A injection for facial rejuvenation.Methods:From October 2018 to September 2019, 14 patients treated with autologous fat filling and botulinum toxin A injection were analyzed retrospectively. The fat granule was harvested by liposuction from waist abdomen or lateral thigh under intravenous anesthesia and local infiltration anesthesia. After purified, the fat was filled into the forehead, temps, eyebrow, glabella, zygomatic, cheek, nasolabial fold, marionette lines and chin in 14 patients with multiple-layer, multiple-tunnel, multiple-point and low-amount injection with even speed slowly. Botulinum toxin A was injected at the same time, 10~20 u in forehead wrinkles; 8-12 u in glabellar wrinkles; 6-12 u in Crow's feet; 1-4 u in marionette lines and 4 u in depressor muscle.Results:After 3-12 months follow-up, 14 cases achieved the expected effect by injecting once and 1 cases achieved satisfactory results after reoperation. The proportion of all parts of the face was more harmonious, the local depression was corrected, the facial wrinkles were reduced, and the skin texture and color were improved. No serious complications occurred.Conclusions:Autogenous fat filling combined with botulinum toxin A injection can enhance the facial rejuvenation, which is worth spreading in clinical practice.