Delivery of erythropoietin by encapsulated myoblasts in a genetic model of severe anemia

BACKGROUND: Existing animal models of anemia inadequately reflect the hematocrit usually present in chronic renal failure (CRF) patients and do not permit long-term treatment studies. The transgenic mouse strain 134.3LC (Epo-TAg(H)) displays a severe chronic anemia resembling that observed clinically during CRF, while displaying an active, normal life span. This phenotype makes it a particularly interesting mouse model for testing erythropoietin (Epo)-based gene transfer strategies. METHODS: Ex vivo gene therapy was employed to administer mouse Epo to homozygous anemic Epo-TAg(H) mice. Encapsulated C(2)C(12) myoblasts genetically engineered to secrete 163 IU mouse Epo/10(6) cells/day were subcutaneously transplanted on the dorsal flank of the mice. Efficacy of delivered Epo was monitored by weekly measurements of animal hematocrit. RESULTS: Most treated homozygous Epo-TAg(H) mice displayed only a transient rise in hematocrit before eventually decreasing to levels as low as 3%. Administering the immunosuppressor anti-CD4+ monoclonal antibody (mAb) to homozygous Epo-TAg(H) mice, beginning at the time of implantation, permitted a rise in hematocrit that remained stable at elevated levels in cases of continued immunosuppression. CONCLUSIONS: Mice having the T antigen insertion in both Epo alleles appeared to develop an immune response to the natural mouse Epo delivered by encapsulated cells. By preventing this reaction using immunosuppression, we demonstrate that encapsulated myoblasts can deliver therapeutic doses of mouse Epo systemically and restore hemopoiesis in a genetic model of severe anemia.     

Smad8/BMP2‐engineered mesenchymal stem cells induce accelerated recovery of the biomechanical properties of the achilles tendon

Tendon tissue regeneration is an important goal for orthopedic medicine. We hypothesized that implantation of Smad8/BMP2‐engineered MSCs in a full‐thickness defect of the Achilles tendon (AT) would induce regeneration of tissue with improved biomechanical properties. A 2 mm defect was created in the distal region of murine ATs. The injured tendons were then sutured together or given implants of genetically engineered MSCs (GE group), non‐engineered MSCs (CH3 group), or fibrin gel containing no cells (FG group). Three weeks later the mice were killed, and their healing tendons were excised and processed for histological or biomechanical analysis. A biomechanical analysis showed that tendons that received implants of genetically engineered MSCs had the highest effective stiffness (>70% greater than natural healing, p < 0.001) and elastic modulus. There were no significant differences in either ultimate load or maximum stress among the treatment groups. Histological analysis revealed a tendon‐like structure with elongated cells mainly in the GE group. ATs that had been implanted with Smad8/BMP2‐engineered stem cells displayed a better material distribution and functional recovery than control groups. While additional study is required to determine long‐term effects of GE MSCs on tendon healing, we conclude that genetically engineered MSCs may be a promising therapeutic tool for accelerating short‐term functional recovery in the treatment of tendon injuries. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1932–1939, 2012     

Effect of anemia and renal cytokine production on erythropoietin production during blood-stage malaria

BACKGROUND: Renal dysfunction and severe anemia are clinical complications of blood-stage malaria. Erythropoietin (Epo) is a hormone produced by the kidney and plays an essential role in stimulating erythrocyte production. Renal dysfunction in malaria is associated with changes in renal cytokine levels, which may affect the production of Epo and the alleviation of anemia. METHODS: Resistant C57BL/6 (B6) and susceptible A/J mice were infected with Plasmodium chabaudi AS. The levels of Epo and cytokines were measured by enzyme-linked immunosorbent assay (ELISA) and the degree of anemia was determined by hematocrit. Regression analyses were employed to estimate the influences of anemia and renal cytokines on the production of Epo during infection. RESULTS: A/J mice developed higher peak parasitemia, more severe anemia, and succumbed as compared to B6 mice, which survived the infection. B6 mice had higher levels of renal tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-10, whereas A/J mice had higher levels of IL-12p70, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, and Epo. Regression analyses revealed that kidney Epo levels were influenced most strongly by changes in hematocrit levels. In addition, albeit to a much weaker degree, kidney Epo levels correlated negatively with GM-CSF levels but positively with IL-10 levels. CONCLUSION: Blood-stage malaria infection modulates the production of renal pro- and anti-inflammatory cytokines in resistant versus susceptible strains of mice differentially. However, despite the fluctuations of renal cytokines, the degree of anemia is the main determinant for Epo production during blood-stage malaria while kidney cytokines may exert secondary influences.     

The effects of ACE inhibitor treatment and ACE gene polymorphism on erythropoiesis in chronic hemodialysis patients

Aggravation of anemia in chronic renal failure patients by angiotensin-converting enzyme inhibitors (ACEIs) has been attributed to the inhibition of angiotensin II which facilitates erythropoietin(Epo) production. This study was aimed at evaluating whether ACEIs aggravate anemia in maintenance hemodialysis patients and to investigate the influence of ACE gene polymorphism on erythropoiesis in these patients. Ninety-one hemodialysis patients were divided into 2 groups, based on whether or not they were administered ACEIs, into the ACEI group(n = 24) and the non-ACEI group(n = 67), and comparisons were made of the doses of recombinant human Epo(rHuEpo) administered, the hematocrit(Hct) and the plasma Epo concentrations. Among the patients in the non-ACEI group, only 17 did not receive rHuEpo, while all of the patients in the ACEI group received rHuEpo. The average dose of rHuEpo was 102.7 +/- 45.4 IU/kg/week in the ACEI group and 57.8 +/- 55 IU/kg/week in the non-ACEI group and the difference between the two groups was statistically significant. A statistically significant difference in the Hct was also observed between the two groups: the mean Hct in the ACEI group was 28.7 +/- 2.9% while that in the non-ACEI group was 31.1 +/- 3.7%. The plasma Epo concentrations were significantly lower in the ACEI group than in the non-ACEI group. No significant differences in the rHuEpo dose and Hct were observed between the three ACE genotype classes in either the ACEI or the non-ACEI group, however, there was a significant difference among the three genotypes in the non-ACEI group in regard to the plasma Epo concentrations; patients with the DD genotype had higher concentrations than those with the DI or II genotypes. These data suggest that anemia in maintenance hemodialysis patients is worsened by ACEIs as a result of the suppression of Epo production. Although it has been suggested that the endogenous Epo concentrations in maintenance hemodialysis patients are associated with ACE gene polymorphism, no significant influence of the ACE genotype on the rHuEpo dose or Hct was evident. Therefore, it is possible that exacerbation of anemia by ACEIs in the patients receiving rHuEpo is a result of an inhibited bone marrow response to Epo.     

Colony-promoting activity in mice kidneys with phenylhydrazine hemolytic anemia

Aqueous anemic mice kidney extracts (MKE) were assessed colony-promoting activity (CPA) of hematopoietic progenitor cells in serum-free cultures stimulated by interleukin-3 and erythropoietin (Epo). Mice with hemolytic anemia followed by phenylhydorazine (PHZ) injection for 3 days showed a decrease in the hematocrit (25.4%) and an increase in serum Epo by 14-fold of the control on day 3 after the treatment. At 3 days, the total number of hematopoietic progenitor cells in the bone marrow of PHZ mice decreased by 67% of the control, while these cells in the spleen increased to 22-fold of the control on day 3 and 55-fold on day 6. A significant increase in CPA was observed in MKE prepared from PHZ mice kidneys. Additionally, bone marrow suppressive anemia induced by 5-fluorouracil resulted in enhanced CPA the same as for PHZ mice, but in contrast, anemia with suppression of Epo-production due to nephrotoxicity induced by cisplatin caused a decrease in CPA. These results suggest that CPA in MKE correlates with hematopoietic conditions, and may have a definite role in hematopoiesis through the function of the kidney.     

Colony-Promoting Activity in Mice Kidneys with Phenylhydrazine Hemolytic Anemia

Aqueous anemic mice kidney extracts (MKE) were assessed colony-promoting activity (CPA) of hematopoietic progenitor cells in serum-free cultures stimulated bv interleukin-3 and erythropoietin (Epo). Mice with hemolytic anemia followed by phenylhydorazine (PHZ) injection for 3 days showed a decrease in the hematocrit (25.4%) and an increase in serum Epo by 14-fold of the control on day 3 after the treatment. At 3 days, the total number of hematopoietic progenitor cells in the bone marrow of PHZ mice decreased by 67% of the control, while these cells in the spleen increased to 22-fold of the control on day 3 and 55-fold on day 6. A significant increase in CPA was observed in MKE prepared from PHZ mice kidneys. Additionally, bone marrow suppressive anemia induced by 5-fluorouracil resulted in enhanced CPA the same as for PHZ mice, but in contrast, anemia with suppression of Epo-production due to nephrotoxicity induced by cisplatin caused a decrease in CPA. These results suggest that CPA in MKE correlates with hematopoietic conditions, and may have a definite role in hematopoiesis through the function of the kidney.     

Prolongation of survival of mice with glioma treated with semiallogeneic fibroblasts secreting interleukin-2.

OBJECT: The current prognosis for patients with malignant brain tumors remains poor, and new therapeutic options are urgently needed. We previously have shown that prolongation of survival can be achieved in C57BL/6 mice (H-2b) with a syngeneic intracerebral or subcutaneous glioma when treated with allogeneic mouse fibroblasts (H-2k) genetically engineered to secrete interleukin-2 (IL-2). Like other antigens, tumor-associated antigens are recognized by cytotoxic T lymphocytes in the context of determinants specified by the major histocompatibility complex class I locus. Because the rejection of allogeneic major histocompatibility complex determinants has the property of an immune adjuvant, immunotherapy of glioma with a cellular immunogen that combines the expression of both syngeneic class I determinants and allogeneic antigens could have advantages over an immunogen that expresses syngeneic or allogeneic determinants alone. METHODS: To investigate this question in a mouse glioma model, we further modified allogeneic mouse fibroblasts (H-2k), not only for IL-2 secretion, but also for the expression of H-2Kb class I determinants. We tested the immunotherapeutic properties of these semiallogeneic cells (LM-IL-2/H-2Kb) in C57BL/6 mice with Gl261 glioma in both subcutaneous and intracerebral glioma models. RESULTS: C57BL/6 mice with either a subcutaneous or intracerebral glioma treated solely by injection of these IL-2-secreting semiallogeneic cells had significantly prolonged survival rates compared with untreated mice or mice treated with cells secreting only IL-2 or cells lacking the H-2Kb determinants. In some instances, the mice treated with the semiallogeneic cells survived indefinitely, suggesting total eradication of the glioma. When a 51Cr release assay was used, the specific immunocytotoxicity measured by release of isotopes from labeled Gl261 glioma cells coincubated with spleen cells from mice immunized with the semiallogeneic IL-2-secreting cells was significantly higher than that of spleen cells from nonimmunized mice or mice immunized with allogeneic cells lacking syngeneic major histocompatibility complex determinants. In addition, antibody depletion studies using monoclonal antibodies against CD8+ and natural killer/lymphokine-activated killer cells demonstrated a specific CD8+ immunocytotoxic response in animals immunized with the semiallogeneic IL-2-secreting cells compared with only a natural killer/lymphokine-activated killer response in mice immunized with the allogeneic IL-2-secreting cells. CONCLUSION: The augmented immune response against glioma in mice treated with the semiallogeneic IL-2-secreting cells points toward a new form of immunotherapy, "immuno-gene therapy," for patients with malignant intracerebral glioma.     

The effect of i.v. iron alone or in combination with low-dose erythropoietin in the rapid correction of anemia of chronic renal failure in the predialysis period

BACKGROUND: It is now more and more evident that anemia of predialysis chronic renal failure (CRF) should be actively treated, since long-standing anemia may cause irremediable damage to the heart. The most common form of treatment of this anemia is subcutaneous erythropoietin (EPO). iron (Fe) deficiency can also contribute to anemia in predialysis CRF, and intravenous iron (i.v. Fe) can frequently improve it. It is possible, therefore, that the combination of EPO and i.v. Fe may have an additive effect, and cause a rapid improvement in anemia with relatively small doses of EPO. PURPOSE: The purpose of this study was an initial study: to assess the ability of a combination of low-dose EPO and i.v. Fe, given weekly for 5 doses, to correct the anemia of predialysis CRF patients compared to the use of i.v. Fe alone in a randomized study. In the follow-up study: to assess the ability of the maintenance of adequate iron stores for one year to achieve and maintain the target Hct of 35% with the minimum dose of EPO. Initial study: METHOD: Ninety predialysis CRF patients (creatinine clearance 10-40 ml/min/1.73 m2 received either: Group A (45 patients): 200 mg i.v. Fe as Fe sucrose (Venofer, Vifor Int.) once per week for 5 doses in combination with 2,000 international units (IU) EPO (Eprex, Cilag-Janssen), subcutaneously given simultaneously also for 5 doses. Group B (45 patients): the same dose of i.v. Fe as in Group A but without EPO. RESULTS: The mean increase in hematocrit (Hct) and hemoglobin (Hb) by one week after the last dose was greater in group A, 4.54 +/- 2.64% (p < 0.01) and 1.37 +/- 0.84 g% (p < 0.01), respectively, than in Group B, 2.74 +/- 2.72% (p < 0.05) and 0.91 +/- 0.78 g% (p < 0.05), respectively. 80% of those in Group A had an increase in Hct of 3 vol% or more compared to 48.9% in Group B (p < 0.01). 40% of those in Group A reached the target Hct of 35% compared to 28.9% in Group B (p > 0.05). Follow-up study: During a 12-month follow-up period, enough i.v. iron was given to maintain the Hct at 35%, while keeping the serum ferritin at < 400 ug/l and % Fe Sat at < 40%. If the i.v. Fe alone was not capable of maintaining the target Hct, EPO was given in increasing doses. Eighteen patients required dialysis. Of the 72 patients who did not require dialysis, 24 (33.3%) maintained the target Hct with i.v. Fe alone, without EPO. All the remaining 48 patients (66.7%) continued to receive EPO in addition to the i.v. Fe, and 47 achieved and maintained the target Hct with a mean EPO dose of 2,979 +/- 1,326 IU/week. CONCLUSION: The combination of low-dose EPO and i.v. Fe had a rapid and additive effect on the correction of anemia in CRF predialysis patients. Maintaining adequate iron stores with i.v. Fe during a subsequent maintenance phase allowed the target Hct of 35% to be reached and maintained with low-dose EPO in two-thirds of the predialysis patients and with no EPO at all in one-third.     

The anemia of end-stage renal disease: hematopoietic progenitor cell response

Patients with the anemia of end-stage renal disease (ESRD) fail to display an appropriate compensatory increase in red cell production. In order to investigate the extent to which the impaired erythropoietic response is determined at the progenitor cell level, we determined the frequencies of marrow colony-forming cells in 11 anemic and 3 non-anemic, dialysis-dependent ESRD patients and 10 healthy individuals. In addition, we measured serum levels of erythropoietin (Epo) by radioimmunoassay. There were no significant differences (P greater than 0.1) between normal and ESRD groups in the frequencies of primitive or late erythroid (BFU-E and CFU-E, respectively), granulocyte-macrophage, and megakaryocyte progenitors, CFU-E/BFU-E ratios, or serum Epo levels. In contrast, 5 non-uremic patients with chronic anemia comparable in severity to the anemic ESRD patients had serum Epo levels and CFU-E/BFU-E ratios that were significantly increased (P less than 0.05 and P less than 0.001, respectively) in comparison to the normal controls and ESRD patients. Pre-dialysis serum and plasma from both ESRD groups were as supportive of autologous erythroid and non-erythroid colony growth in vitro as normal serum and plasma; inhibition was not observed. We conclude that the relative numbers of erythroid and non-erythroid progenitors and the majority of serum Epo levels are unchanged from normal in patients with the anemia of ESRD. However, their normal CFU-E/BFU-E ratio reflects an inadequate compensatory erythropoietic response due to their inability to appropriately increase Epo production in response to anemia. Inhibitors of autologous erythroid colony formation were not detected in ESRD serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

间充质干细胞移植抑制小鼠急性移植物抗宿主病的实验研究(英文)

目的研究间充质干细胞的体内免疫调节活性。方法将C57/BL脾脏细胞输注入受亚致死量照射的BALB/c小鼠,建立急性移植物抗宿主病模型,受体小鼠或同时接受密质骨骨髓来源的间充质干细胞,流式细胞技术检测脾细胞表面表达的活性分子。结果模型经输注间充质干细胞后,鼠脾脏CD11b+细胞表达的CD86和CD69减少,提示抗原呈递细胞的成熟度下降。此外,作为一种效应T细胞早期活化和发育的标记,CD3+CD69+细胞/CD3+细胞的比值降低,导致脾内CD3+细胞的绝对和相对数目减少。但是,在同系脾细胞输注模型中,CD11b+细胞表面的CD69和MHCⅡ表达以及CD3+细胞表面的CD69不受影响。结论 MSCs可以分3个阶段抑制急性移植物抗宿主病,有望成为急性移植物抗宿主病的有效治疗手段,但仍需进行深入研究。     

生血肾灵治疗肾性贫血的临床研究

目的：观察生血肾灵对肾性贫血的疗效和机制。方法：收集31例慢性肾衰竭（CRF）处于失代偿期和肾功能衰竭期的患,给予生血肾灵治疗2个月,观察患的一般症状和体征,并于治疗前后分别测定病人的血色素（Hb)、红细胞压积（Hct)、血肌酐（Scr)、血尿素氮（BUN）、中分子物质（MMS）、血红细胞超氧化物歧化酶（SOD）活性和脂质过氧化物（LPO）,并进行治疗前后的对比。结果：经生血肾灵治疗后患的症状和体征明显改善;治疗后的Hb、Hct及SOD较治疗前明显升高（P＜0．01）;Scr、BUN、MMS及LPO较治疗前显降低（P＜0．01）。结论：生血肾灵能够明显改善CRF患的症状和体征,延缓肾衰进程,纠正肾性贫血。     

The hemodynamic effects of halothane in anemic dogs

Hemodynamic dose-response curves and MAC, apneic and lethal concentrations for halothane were determined in two groups of dogs at a normal hematocrit (Hct) (39 +/- 6), and during acute anemia (n = 4) and subacute anemia (n = 4), with a combined Hct of 18 +/- 6. The ratio of subendocardial/subepicardial blood flow was determined by the injection of radioactive microspheres. MAC, apneic, and lethal concentrations of halothane were not significantly different in anemic animals. There were also no differences in the halothane dose-response curves plotted for cardiac output, blood pressure, heart rate, left ventricular end-diastolic pressure, or arterial base excess. In 32 microsphere injections with Hct as low as 13, no dog had redistribution of myocardial blood flow suggestive of subendocardial ischemia. We conclude that halothane is a safe anesthetic during severe anemia.     

Dose response to a single intramuscular injection of recombinant adeno-associated virus-erythropoietin in monkeys

BACKGROUND: Anemia is a significant problem in many disease states. Erythropoietin (Epo) has been used in the treatment of anemia associated with numerous chronic diseases. This study investigates the dose-response profiles of a single intramuscular (im) injection of a recombinant adeno-associated virus vector (rAAV) containing the Epo gene with the goal of achieving a sustained elevation of hematocrit (Hct). METHODS: Cynomolgus (cm) monkeys were given single injections of different doses of rAAV-cm-Epo. The biological effect of Epo gene expression was monitored by determining the Hct levels and circulating hormone levels by ELISA. Antibody to the rAAV capsid protein was also measured over the 41-week period of the experiment. RESULTS: Epo expression was noted only when 2 x 10(11) or more particles were injected. Epo was noted to be increased as soon as 1 week postinjection and was maximum in 6 to 8 weeks. This level of expression remained constant for nearly 20 weeks. Animals given the highest dose of rAAV developed a higher Hct over the first 8 weeks postinjection than those given an intermediate dose. However, the maximum levels of hemoglobin were the same. There was a weak correlation between amount of rAAV injected and capsid antibody response. CONCLUSIONS: AAV vectors are able to transduce skeletal muscle and are capable of achieving sustained expression and systemic delivery of a therapeutic protein following a single im administration. Dose responses to rAAV-Epo are achievable, although a threshold inoculum of virus is necessary to produce an effect and the therapeutic window is narrow.     

Beneficial influence of recombinant human erythropoietin therapy on the rate of progression of chronic renal failure in predialysis patients.

BACKGROUND: Partial correction of anaemia with recombinant human erythropoietin (rHuEpo) has been shown to markedly improve the general condition and quality of life of predialysis patients, but the effects of rHuEpo therapy on blood pressure and the rate of progression of chronic renal failure (CRF) are still disputed. In particular, no study evaluated the time duration until the start of maintenance dialysis in treated patients, compared to untreated predialysis patients. METHODS: We retrospectively evaluated the rate of decline of creatinine clearance (Delta Ccr) and the duration of the predialysis period in 20 patients with advanced CRF treated with rHuEpo (Epo+ group), and in 43 patients with a similar degree of CRF but with less marked, asymptomatic anaemia, not requiring rHuEpo therapy (Epo- group). All patients were submitted to identical clinical and laboratory surveillance. All received similar oral supplementation with B(6), B(9), and B(12) vitamins and oral iron supplementation. Maintenance dose of subcutaneous epoetin was 54.3+/-16.5 U/kg/week (median dose 3300 U/week). RESULTS: Initial and final haemoglobin (Hb) levels were 8.8+/-0.7 and 11.3+/-0.9 g/dl in the Epo+ group, vs 10.9+/-1.2 and 9.5+/-0.9 g/dl in the Epo- group. In the Epo+ group, Delta Ccr declined from 0.36+/-0.16 during the preceding 24 months to 0.26+/-0.15 ml/min/ 1.73 m(2)/month after the start of rHuEpo therapy (P<0.05). No significant variation was observed in the Epo- group. Time duration until the start of dialysis was 16.2+/-11.9 in the Epo+ group, compared to 10.6+/-6.1 months in the Epo- group (P<0.01). Slowing of progression was observed in 10 Epo+ patients, whereas no significant variation in Delta Ccr occurred in the other 10. There was no difference in previous Delta Ccr rate, nor in Hb or blood pressure levels while on rHuEpo therapy between the two subgroups. CONCLUSIONS: Our study affords conclusive evidence that rHuEpo therapy did not result in accelerated progression of CRF in any treated predialysis patients, nor deleterious increase in blood pressure, but instead resulted in significant slowing of progression and substantial retardation of maintenance dialysis. Such encouraging results remain to be validated in a large prospective, randomized study.     

Cerebral blood flow and vasodilatory capacity in anemia secondary to chronic renal failure

BACKGROUND: Our previous study reported that cerebral oxygen extraction fraction (OEF) increased in hemodialysis patients with anemia. The increased OEF suggests that the cerebral vasodilatory capacity might be impaired in these patients. To clarify this issue, we measured the CO2 response in patients with anemia secondary to chronic renal failure (CRF) using positron emission tomography (PET). METHODS: Ten anemic patients with CRF (6 females and 4 males) and 6 age-matched normal controls were studied. The underlying diseases of CRF were glomerulonephritis in 8 patients, systemic lupus erythematosus (SLE) in one patient, and hypertension in one patient; in this cohort, 5 patients were on hemodialysis treatment and the remaining 5 patients were in a pre-hemodialysis state. The cerebral blood flow (CBF) was measured by the O-15 H2O bolus injection method with each patient in a resting state and during 5% CO2 inhalation. The CO2 response was estimated as the percentage change of CBF per 1 mm Hg change of PaCO2. RESULTS: The CO2 response was significantly attenuated in anemic patients with CRF in comparison to the normal controls, and it inversely correlated with the severity of anemia. There was no significant difference in the CO2 response between the hemodialysis and pre-hemodialysis patients. The CO2 response significantly correlated with CBF and the cerebral metabolic rate for oxygen (CMRO2) at rest, however, it did not correlate with OEF and cerebral blood volume (CBV). CONCLUSIONS: The present study revealed the existence of a reduced cerebral vasodilatory capacity in anemic patients with CRF, suggesting that chronic hypoxic brain damage might play a role in the impaired cerebrovascular response to CO2.     

MSCs与CD34~+单核细胞共培养联合脱细胞支架植入体内构建兔阴茎海绵体

目的研究将异体骨髓间充质干细胞(MSCs)和CD34~+单核细胞(MNCs)共培养体系种植于脱细胞海绵体支架上在兔体内构建阴茎海绵体组织的可行性。方法分别从兔骨髓与兔外周血中提取并鉴定MSCs与CD34~+MNCs,将两种细胞组分以107/mL的密度种植于制备好的兔阴茎海绵体脱细胞胶原支架上,同时将相同密度的MSCs、CD34~+MNCs分别种植于支架上,培养4天后植入一种兔阴茎海绵体缺损模型中。3个月后对工程化组织进行取材,行HE及Masson三色染色等组织学检测。结果 MSCs和CD34~+MNCs共培养组近白膜处的海绵体保留了正常海绵体结构,而MSCs组、CD34+单核细胞组及空白支架组以疤痕增生为主,尽管较之CD34~+单核细胞组及空白支架组,间充质干细胞组疤痕组织中的血管数目较多(P0.05)。结论我们的研究证明了将MSCs和CD34~+MNCs的共培养体系种植于脱细胞支架上植入兔体内可以构建出一段组织学上与原生海绵体相似的组织;CD34~+MNCs在体内主要通过协同MSCs而非单独发挥其促血管化的生物学效应。     

同种肾组织移植治疗慢性肾功能衰竭性贫血的实验研究

以Ｗｉｓｔａｒ雄性大鼠为受体，建立慢性肾功能衰竭动物模型，将鼠婴肾组织声多点植入受体双侧后肢皮下和筋膜下。结果表明，３０天后移植物的体积由１ｍｍ＾３增至４ｍｍ＾３大小，表面血管网丰富；光镜下见肾小球、肾小管结构正常。促红细胞生成素（ＥＰＯ）着色颗粒主要分布在肾小球区，移植组着色程度明显增高。血红蛋白和４促红细胞生成素随移植的时间延长而逐渐升高，实验结果提示，此方法有可能为治疗慢性肾功能衰竭性贫血提     

Temporal relationship of tissue somatostatin-like immunoreactivity to metabolic changes in genetically obese and diabetic mice

Somatostatin-like immunoreactivity (SRIF-LI) content in 2 N acetic acid extracts of hypothalamus, gastric antrum, and pancreas was measured in genetically obese (C57BL/6J ob/ob and db/db) and diabetic (C57BL/KsJ db/db and ob/ob) mice and normal littermate controls from 5 to 24 wk to determine the relationship of previously reported changes to the development of metabolic abnormalities. Hypothalamic SRIF-L concentration was similar in control, diabetic, and obese mice at all ages and increased progressively with age in all groups. Gastric antrum SRIF-LI was similar in all groups of mice at all ages. Obese mice gained weight progressively and showed moderate hyperglycemia and marked hyperinsulinemia from 5 wk of age. Pancreatic SRIF-LI content in obese (C57BL/6J) animals was similar to that in lean littermate controls, but pancreatic SRIF-LI concentration (expressed by weight or protein content) was decreased until 8 (6J ob/ob) and 10 (6J db/db) wk. Diabetic (C57BL/KsJ) mice showed a similar metabolic pattern until 10 wk with no change in pancreatic SRIF-LI content or concentration. Thereafter a progressive fall in serum insulin and a marked rise in serum glucose was associated with increasing pancreatic SRIF-LI content and concentration. These studies suggest that the genetically hyperphagic syndromes are unassociated with any change in hypothalamic or gastric SRIF-LI; that pancreatic SRIF-LI increases occur in response to, rather than as the cause of, relative hypoinsulinemia; and that the genetic background of the mice (KsJ or 6J) rather than the mutant gene (db or ob) determines the defect in carbohydrate metabolism and the pancreatic SRIF-LI response.     

Immunogene therapy with interleukin-2-secreting fibroblasts for intracerebrally metastasizing breast cancer in mice

OBJECT: Breast cancer is the second leading cause of cancer-related death in American women. Brain metastases occur in 15 to 30% of patients with breast cancer, and this usually results in death. Despite the availability of surgery, radiotherapy, and chemotherapy, the prognosis for patients with breast cancer that has metastasized to the intracerebral region remains poor. This study was designed to determine if an intracerebrally metastasizing breast tumor could be treated successfully with a cellular vaccine consisting of allogeneic fibroblasts (H-2K) modified to secrete interleukin (IL)-2. METHODS: The authors used EO771 breast cancer cells, derived from a spontaneously arising breast-cancer tumor in C57BL/6 mice. The authors first determined the length of survival of C57BL/6 mice that had been injected with varying numbers of EO771 cells into the right frontal lobes and found that 100% of those animals that received a dose of 10(4) cells died within 41 days, whereas 100% of the group that received 10(3) cells died within 23 days. Based on these results, 5 x 10(4) EO771 cells were injected into the right frontal lobe of C57BL/6 mice and the animals were treated intracerebrally with a single intratumoral injection of 10(6) allogeneic fibroblasts genetically engineered to secrete IL-2. The allogeneic fibroblasts transfected with the IL-2 gene formed large quantities of IL-2 as measured by an enzyme-linked immunosorbent assay. Control groups of animals were treated with either allogeneic fibroblasts transfected with the retroviral vector, but not the IL-2 gene, or with media. The effects of this treatment on the animal's survival and the tumor's histopathological characteristics were investigated. The results indicate a significant prolongation (p < 0.005) of survival in animals with intracerebrally metastasizing breast cancer treated only with IL-2-secreting allogeneic fibroblasts. Tumor did not develop in four of 10 animals in the IL-2-treated group, and these animals were rechallenged at 90 days by intracerebral injection of tumor, but no treatment cells, and compared with four naive animals receiving intracerebral tumor. Again, animals that previously had been treated with IL-2-secreting fibroblasts had a markedly prolonged survival (p < 0.05) compared with control animals following a second challenge with tumor cells. Histopathological examination revealed smaller tumors associated with lymphocytic infiltrations in the treated animals. CONCLUSIONS: This work represents a new treatment for breast cancer that has metastasized to the brain in which a cellular vaccine consisting of allogeneic fibroblasts genetically engineered to secrete cytokines is used as a novel means for delivery of immunogene therapy and demonstrates the induction of long-term immunity against tumor.